Rotaviruses.

Virus Research 190 (2014) 75–96

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Virus Research journal homepage: www.elsevier.com/locate/virusres

Review

Rotaviruses Ulrich Desselberger ∗ Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK

a r t i c l e

i n f o

Article history: Received 8 May 2014 Received in revised form 26 June 2014 Accepted 26 June 2014 Available online 9 July 2014 Keywords: Rotavirus Structure–function studies Replication cycle Pathogenesis Immune responses and correlates of protection Prevention by vaccination

a b s t r a c t Recent advances of rotavirus (RV) basic and applied research are reviewed. They consist of determination of the RV particle structure and functions of structural proteins, classification into genotypes based on whole genome analyses, description of the RV genome and gene protein assignments, description of the viral replication cycle and of functions of RV-encoded non-structural proteins as well as cellular proteins and cellular organelles involved, the present status of RV genetics and reverse genetics, molecular determinants of pathogenesis and pathophysiology, the RV-specific humoral and cell-mediated immune responses, innate immune responses and correlates of protection, laboratory diagnosis, differential diagnosis and present status of treatment, the molecular epidemiology and mechanisms of evolution of RVs, the development and universal application of RV vaccines as well as issues arising from present universal RV vaccination programs and work on alternative vaccines. The review concludes by presenting problems requiring further exploration and perspectives of future basic and translational research. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The rotavirus genome and gene-protein assignments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The rotavirus replication cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Virus particle attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Virus particle penetration and uncoating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. RV plus strand (m)RNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Viroplasm formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. RNA packaging, minus strand RNA synthesis and DLP formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Virus particle (virion) maturation and release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Persistent RV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Growth of RVs in stem cell organoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9. Cellular proteins involved in RV replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. RV genetics and reverse genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Molecular pathogenesis and pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Immune responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. RV-specific humoral and cell-mediated immune responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Innate immune responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Correlates of protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Clinical symptoms and differential diagnosis, laboratory diagnosis and treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Molecular epidemiology and mechanisms of evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Prevention: development and universal application of RV vaccines, issues arising from present RV vaccination programs . . . . . . . . . . . . . . . . . . . . . 12. Perspectives of future basic and translational research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

76 76 76 78 78 78 79 79 80 81 81 81 81 81 82 82 83 83 84 84 84 85 86 87

∗ Tel.: +44 1223 763403; fax: +44 1223 336846. E-mail address: [email protected] http://dx.doi.org/10.1016/j.virusres.2014.06.016 0168-1702/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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U. Desselberger / Virus Research 190 (2014) 75–96

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Rotaviruses (RVs) were recognized as a major cause of acute gastroenteritis (AGE) in infants and young children in 1973 (Bishop et al., 1973; Flewett et al., 1973). Before that year RVs had already been discovered in diarrheic mice (Adams and Kraft, 1963), monkeys (Malherbe and Harwin, 1963) and cattle (Mebus et al., 1969) and since then in the young of many mammalian species including bats (Estes and Greenberg, 2013; He et al., 2013) and also in birds (Kindler et al., 2013; Otto et al., 2012; Trojnar et al., 2009). During the last 40 years an enormous amount of basic research on RV structure, replication, pathogenesis and immune responses has accumulated. Due to modern diagnostic techniques the molecular epidemiology of RVs has been extensively explored. Recently, two live attenuated RV vaccines have been licensed in many countries and are increasingly being applied in universal vaccination programs. In the following I will attempt to give an up-to-date review of important recent achievements in research and prevention and to formulate remaining open questions and perspectives.

2. Structure The fully infectious RV particle (=virion) consists of 3 protein layers and is also termed triple-layered particle (TLP). By electron microscopy, TLPs resemble wheels (lat. rota), and this appearance has led to the name of Rotavirus for the genus (Flewett et al., 1974). Based on cryo-electron microscopy and image reconstruction data (reviewed by Jayaram et al., 2004), the following structure of icosahedral symmetry has been recognized: the single layered particle (SLP = core shell) is formed by 120 molecules of the viral protein 2 (VP2), arranged as 60 dimers in a T = 1 symmetry (Fig. 1C). Five of the dimers form a decamer around the fivefold symmetry axis, and 12 decamers make up the core protein layer which is uniform except for small pores along the fivefold axis (McClain et al., 2010). A ‘fivefold hub’ (density) projecting into the core interior along the fivefold axis was first thought to be contributed by the N-terminus of VP2 (McClain et al., 2010), but was more recently recognized to result from the VP1 structure (Estrozi et al., 2013). Replication enzyme complexes, consisting of VP1 and VP3, are located at the inside of the core at the axis of fivefold symmetry (Fig. 1C and D) opposite the class I channels (McClain et al., 2010; Trask et al., 2012a, 2012b; Estrozi et al., 2013) and are in intense contact with one particular dedicated genomic dsRNA segment via VP1 (Periz et al., 2013). The core shell encloses the viral genome of 11 segments of dsRNA as well as the viral RNA dependent RNA polymerase (RdRp), VP1 and the capping enzyme, VP3. The genomic RNA segments have been proposed to form conical cylinders around the replication complexes (Prasad et al., 1996; Jayaram et al., 2004) (Fig. 1D) but details of the dsRNA structure within the core are just beginning to be explored. Thus, in actively transcribing double-layered particles (DLPs, see below), a decreased order of the middle (VP6) layer was found to be accompanied by increased order of the core content (Kam et al., 2014). Similar partial order of dsRNA segments was observed in core particles of bluetongue virus (Gouet et al., 1999). The 11 RNA segments (Fig. 1A) have very short completely conserved 5 and 3 terminal nucleotide (nt) sequences, 5 -GGC · · · ACC-3 . The untranslated regions (UTR) of the RNA segments (+ sense) are small: 9–48 nt at the 5 end, and 17–182 nt at the 3 end. The 5 and 3 ends are of partial inverted complementarity and are subject to long range

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interactions (LRI) (Tortorici et al., 2006; Li et al., 2010). Those at the 3 end function as RdRp recognition signals (Tortorici et al., 2003) and also interact with NSP3 (Chizhikov and Patton, 2000; Vende et al., 2000), involved in activation of translation (see below). The RdRp structure has been solved at a resolution of 2.9 A˚ (Lu et al., 2008) (Fig. 2): VP1 forms a cage-like structure disrupted by 4 tunnels (leading to the catalytic site in the center of the molecule) which are proposed to allow for: (1) the entry of free nucleoside triphosphates (NTPs), (2) the entry of template ssRNA, (3) the exit of the (+) ssRNA product, and (4) the exit of (−) ssRNA (transcription) or dsRNA (replication). Tunnel 3 is positioned toward the class I channel inside of VP2 (Settembre et al., 2011; Estrozi et al., 2013), permitting release of (+) RNAs into the cytoplasm; tunnel 4 directs the newly replicated dsRNA toward the core interior. Analysis of different VP1-polyribo-oligonucleotide complexes has resulted in the finding that the 5 -UGUG-3 sequence at the 3 end of the RNA is specifically recognized at the template entry channel (Lu et al., 2008). In many aspects the structure of the RV RdRp is similar to the corresponding enzyme of orthoreovirus (Tao et al., 2002). The viral core is surrounded by 260 trimers of VP6, which form the middle layer and constitute double-layered particles (DLPs) (Fig. 1C and E). The VP6 structure has also been determined (Mathieu et al., 2001): VP6 trimers make contact with both the underlying core (VP2) (Charpilienne et al., 2002) as well as VP7 and VP4 trimers on the outside. The DLPs in turn are covered by 260 trimers of VP7 and 60 spikes of VP4 trimers (=180 molecules) to form the TLPs (Fig. 1B and C). The virions contain 132 channels along the axes of fivefold (12 channels of class I), threefold and twofold symmetry (60 class II and 60 class III channels, respectively) (Jayaram et al., 2004). The three-dimensional (3D) structure of the RV virion has recently been determined by electron cryomicroscopy (cryoEM) and singleparticle tomography at about 4.3 A˚ resolution (Settembre et al., 2011): there is an intense interaction of the VP4 trimer with both VP6 (into which the base of VP5* is half buried) and VP7 (Fig. 3). The structure and interactions of VP4 with other structural RV proteins permit the description of a mechanism of RV entry (see below). It has previously been shown that VP4 trimers can only be observed by cryo-EM in TLPs grown in the presence of trypsin (Crawford et al., 2001). Since the stoichiometry of VP4 in RVs grown in the presence and absence of trypsin is identical, it was concluded that VP4 spikes of RV grown in the absence of trypsin are icosahedrally disordered. By single particle cryo-EM and cryo-electron tomography (cryo-ET) it has recently been demonstrated for 2 RV strains that the VP4 spike structure of RV particles grown in the absence of trypsin is indistinguishable from that of particles grown in the presence of trypsin (Rodríguez et al., 2014), suggesting that the previous lack of observation of VP4 structures in trypsin-free grown RV particles was mainly due to the imposition of icosahedral structure requirements on the previous cryo-EM data and that proteolytic cleavage of VP4 mainly achieves conformational changes enabling viral entry into cells. As an aside, the science of structural biology is closely related to talents in visual arts as noted by Stephen and Baumeister (2008).

3. Classification Rotaviruses constitute the genus Rotavirus, one of the 15 genera of Reoviridae family which is subdivided into the sub-families of


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Fig. 1. Aspects of rotavirus structure. (A) PAGE gel showing 11 dsRNA segments comprising the rotavirus (RVA) genome. The gene segments are numbered on the left and the proteins they encode are indicated on the right. (B) Cryo-EM reconstruction of the rotavirus triple-layered particle. The spike protein VP4 is colored in orange and the outermost VP7 layer in yellow. (C) A cutaway view of the rotavirus TLP showing the inner VP6 (blue) and VP2 (green) layers and the transcriptional enzymes (in red) anchored to the inside of the VP2 layer at the fivefold axes. (D) Schematic depiction of genome organization in rotavirus. The genome segments are represented as inverted conical spirals surrounding the transcription enzymes (shown as red balls) inside the VP2 layer in green. (E and F) Model from Cryo-EM reconstruction of transcribing DLPs. The endogenous transcription results in the simultaneous release of the transcribed mRNAs from channels located at the fivefold vertices of the icosahedral DLP. From Jayaram et al. (2004). With permission of the authors and the publisher.

Fig. 2. Structure of the RV RdRp VP1. Surface rendering (A) of the complete VP1 polypeptide chain. The N-terminal domain is in yellow, the C-terminal (bracelet) domain is in pink, and the C-terminal plug is in cyan. (B) Sagittal cutaway of the image in (A), after rotation to the left by 90◦ , showing the four tunnels extending into the central cavity. From Lu et al. (2008). With permission of the authors and the publisher.

the Sedoreovirinae (genera Cardoreovirus, Mimoreovirus, Orbivirus, Phytoreovirus, Rotavirus, Seadornavirus) and the Spinareovirinae (genera Aquareovirus, Coltivirus, Cypovirus, Dinovernavirus, Fijivirus, Idnoreovirus, Mycoreovirus, Orthoreovirus, Oryzavirus). According to the serological reactivity and genetic variability of VP6, at least 8 different groups, also termed species, are differentiated (termed RVA-RVH) (Matthijnssens et al., 2012). The RVA species comprises at least 27 G types (according to the nt sequence of VP7) and 37 P types (according to the nt sequence of VP4) (Matthijnssens et al., 2011a; Rotavirus Classification Working Group, 2013). For G types, serotypes and genotypes are synonymous, e.g. G1, G2, etc. For P types, there are many more P genotypes

than reference sera determining P serotypes: therefore, a double nomenclature has been introduced, e.g. P1A[8] designating the P serotype 1A and P genotype 8, etc. (Estes and Greenberg, 2013). A comprehensive, nt sequence-based classification comprising the complete genome has been introduced for RVAs, in which the VP7–VP4–VP6–VP1–VP2–VP3–NSP1–NSP2–NSP3–NSP4–NSP5/6 genotypes are identified and differentiated according to particular cut-off points of nt sequence identities (Matthijnssens et al., 2008a, 2008b, 2011a; Maes et al., 2009). Table 1 demonstrates the enormous degree of genomic diversity among RVAs co-circulating in different population groups and animals at various times. Recently, similar comprehensive genotype differentiations for

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and protein-function assignments have been determined for several RVA strains and are shown for the bovine RVA RF (G6P6[1]) strain in Table 2. Besides the 6 structural proteins described above (VP1, VP2, VP3, VP4, VP6, VP7), 5–6 non-structural proteins (NSP1–NSP5/6) are produced the functions of which will be described below. RVCs do not encode an NSP6. The differences at the 3 end of the RNAs between different RV species are considered to be the reason for their lack of reassortment (see below). The short 5 and 3 UTRs are not long enough to singly contain the putative packaging sequences important for RV morphogenesis, but direct experimental evidence is so far missing due to the lack of a tractable reverse genetics system for RVs (see below). The packaging signals of the RNA segments of influenza viruses and as far as explored of the orbiviruses include sequences of parts of the UTRs and the adjacent ORFs (Essere et al., 2013; Matsuo and Roy, 2009). Rotavirus genes can be ‘rearranged’, due to partial nt duplications or deletions of RNA segments generated by special forms of intragenic recombination (Desselberger, 1996). Rotaviruses with rearrangements in 3 genes were found to package approximately 1800 additional bp of RNA, i.e. about 10% of the genome, to be replication competent and to be physically and genetically stable (Hundley et al., 1987; McIntyre et al., 1987). Genome rearrangements can be experimentally reproduced by serial passage of RVs in cell culture at high MOI (Hundley et al., 1985). 5. The rotavirus replication cycle Fig. 3. Interaction of rotavirus structural proteins. Cutaway view of the interactions of VP4 within the TLP. VP8* is in magenta and VP5* is in red. The VP5* segment that will form the coiled coil (c-c) in the ‘post-entry’ conformation is in cyan. The globular foot domains of the VP5* is anchored at a six-coordinated position in the VP6 layer (green); a VP7 trimer (yellow) caps each trimer of VP6. Both form surface lattices in T = 13 levo icosahedral symmetry. The VP6 lattice overlays the VP2 shell (dark blue), which surrounds the coiled RNA genome (not shown). From Settembre et al. (2011). With permission of the authors and the publisher. Table 1 Genotypes of species A rotaviruses. RV protein

Percent identitya

Number of genotypesb

Genotype (acronym underlined)

VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5

80 80 85 83 84 81 79 85 85 85 91

27G 37P 18I 9R 9C 8M 19A 10N 12T 15E 11H

Glycosylated Protease-sensitive Inner capsid RdRpc Core protein Methyltransferase Interferon Antagonist NTPase Translation enhancer Enterotoxin PHosphoprotein

Updated from Matthijnssens et al. (2008a, 2008b). a Nucleotide percent identity cut off value defining genotypes. b October 2013 (Rotavirus Classification Working Group, 6th meeting, Valencia/ Spain). c RNA-dependent RNA polymerase.

RVBs and RVCs have started to be established (Marthaler et al., 2012, 2013). 4. The rotavirus genome and gene-protein assignments The RVA genome is approximately 18,500 bp in size and consists of 11 segments of dsRNA which encode 6 structural and 6 non-structural proteins. The genes are monocistronic, except for genome segment 11, which encodes two proteins. The gene segments are between 667 and 3302 bp in length. The gene-protein

The RV replication cycle (Fig. 4) includes the following steps: - attachment, mediated by VP4 and VP7 - penetration and uncoating - plus strand ssRNA (=mRNA) synthesis, mediated by VP1, VP3 and VP2 - viroplasm formation, mediating RNA packaging, minus strand RNA synthesis (=RNA replication) and DLP formation - Virus particle maturation (to TLPs) and release 5.1. Virus particle attachment RV attachment is a complex process (López and Arias, 2004). First, the infectious RV particle, the TLP, interacts by its VP4 spikes with cellular receptors (attachment receptors). The receptors contain sialic acid (SA) in terminal or sub-terminal positions. For the rhesus RV VP4 the SA binding site has been structurally identified (Dormitzer et al., 2002a, 2002b). Treatment of cells by neuraminidase abolishes infection by certain RV strains (e.g. SA11) which have therefore been called SA-sensitive strains (Isa et al., 2006). Other strains which can infect neuraminidase-treated cells, have initially been termed ‘SA-resistant’ strains (e.g. Wa and DS-1) but were then shown to bind to receptors containing SA in internal position of glycolipids which are insensitive to neuraminidase treatment, e.g. the GM1 ganglioside (Guo et al., 1999; Haselhorst et al., 2009). On the virus side, attachment is mediated by the VP8* subunit of VP4: a shallow groove of the VP8* surface interacts with SAs on cellular glycans (Dormitzer et al., 2002a, 2002b). More recently it has been demonstrated that certain RV strains, e.g. G10P[11], bind to non-SA containing histo blood group antigens (HBGA) via the Gal␤1–4GlcNAc motif (Hu et al., 2012; Huang et al., 2012; Ramani et al., 2013). For the P[8] genotype it has been shown that its interaction with HBGA receptor depends on a functioning FUT2 enzyme (Imbert-Marcille et al., 2014). Interestingly, certain RVs and noroviruses both use polymorphic HBGA receptors for attachment (Tan and Jiang, 2014; Le Pendu et al., 2014). Various cellular surface molecules can act as (post-attachment) co-receptors,


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Table 2 Gene-protein assignments, protein localization and protein-function assignment adapted to bovine rotavirus RF strain (G6P6[1]). Genome segment

Size (bp)

Encoded protein

Size (kDa)

Location in virion

Molecules/virion

Functions

1 2 3

3302 2687 2592

VP1 VP2 VP3

125 94 88

Core Core Core

12 120 12

4

2362

VP4a

86

Outer layer

180

5 6

1581 1356

NSP1 VP6

58 44

Nonstructural Middle layer

780

7

1062

VP7b

37

Outer layer

780

8

1059

NSP2

36

Nonstructural

9

1074

NSP3

34

Nonstructural

10

751

NSP4

20

Mainly nonstructural

11

666

NSP5

21

Nonstructural

NSP6c

12

Nonstructural

RdRp; ssRNA binding; complex with VP3 Core shell; RNA binding; required for RdRp activity Guanylyltransferase; methyltransferase; 2 ,5 -phosphodiesterase; ssRNA binding; complex with VP1 Homotrimer; P type neutralization antigen; attachment protein; protease enhanced infectivity; virulence; fusion with cell membrane Interferon antagonist; E3 ligase; RNA binding Homotrimer, species determinant; protection (intracellular neutralization); required for transcription Homotrimer; glycoprotein; G type neutralization antigen; Ca2+ dependent Octamer; binds RNA, NTPase; NDP kinase; helix destabilizing; essential for viroplasm formation Dimer; binds to: 3 terminus of viral ss (+) RNA, cellular eIF4G, Hsp90; displaces PABP; inhibits host protein translation RER transmembrane glycoprotein; viroporin; intracellular receptor for DLPs; interacts with viroplasms and autophagy pathway; modulates intracellular Ca2+ and RNA replication; enterotoxin (secreted); virulence Dimer; phospho- and O-glycosylated protein; RNA binding; kinase; essential for viroplasm formation; interaction with VP2 Interaction with NSP5, localized in viroplasm

a b c

Very few

Cleaved by trypsin or cellular protease into VP5* + VP8*. Signal peptide cleaved off. Second ORF of RNA11.

such as several integrins (␣2␤1, ␣␯␤3, ␣x␤2, ␣4␤1), which react with integrin ligand motifs on VP5* or VP7 (motif DGE on VP5* or motifs GPR and CNP on VP7) (Coulson et al., 1997; Graham et al., 2003; Zárate et al., 2004; Gutiérrez et al., 2010), or with the human heat shock cognate protein 70 (Hsc70) (Guerrero et al., 2002; Zárate et al., 2003; Pérez-Vargas et al., 2006). All co-receptors are associated with lipid rafts, i.e. detergent-resistant lipids close to the cellular plasma membrane acting as platforms on which RV TLPs associate (Isa et al., 2004). The relative importance of the various receptors for human RV infection is beginning to be assessed (Fleming et al., 2014). It should be noted that some RV strains hemagglutinate (Kalica et al., 1978; Bastardo and Holmes, 1980) and that it is the VP8* component of VP4 (at the tip of the spikes) which mediates this interaction (Mackow et al., 1989; Fiore et al., 1991). 5.2. Virus particle penetration and uncoating Before trypsin cleavage, VP4 spikes are flexible and thus not visible by EM, although they are present in a virtually identical structure as recently confirmed by single particle cryo-ET (Rodríguez et al., 2014). Upon contact with the cellular receptor, the VP4 spikes of RV TLPs undergo conformational changes in such a way that the lipophilic domains of VP5* which are normally hidden below VP8* are exposed on the surface in form of a ‘post-penetration umbrella’ conformation (Kim et al., 2010; Trask et al., 2010a; Settembre et al., 2011). Treatment of RV particles with trypsin seems to favor this transition (Crawford et al., 2001; Rodríguez et al., 2014) and convey full infectivity to the TLPs (Estes et al., 1981). Partially trypsinresistant RV mutants were shown to have a delay in cellular entry, but then to replicate as well as wildtype (wt) virus (Trask et al., 2013). Following binding, the mechanism of cell penetration of RV particles remains unclear; it may occur by receptor-mediated endocytosis or direct membrane penetration, with solubilization of the outer capsid proteins due to low Ca2+ concentrations in endosomes

(Ludert et al., 1987) to yield DLPs. The presence of cholesterol and of the GTPase dynamin on cell membranes are required for RV entry, but not for all RV strains (Sánchez-San Martín et al., 2004). Genomewide RNAi screening revealed that components of the ‘endosomal sorting complex for transport’ (ESCRT) are involved in RV entry (Silva-Ayala et al., 2013). RV strains differ in the usage of endosomal pathways; some require Rab7-mediated transport to late endosomes (Díaz-Salinas et al., 2014). 5.3. RV plus strand (m)RNA synthesis RV particles possess their own transcription complexes (TCs), consisting of VP1, the viral RNA-dependent RNA polymerase (RdRp), and VP3, the viral capping enzyme (with phosphodiesterase, guanylyltransferase and methylase activities). The TCs are localized at the inner surface of the VP2 (core) layer at the fivefold symmetry axes, i.e. at 11 or 12 vertices (Jayaram et al., 2004). Each TC is complexed with a dedicated viral RNA segment (Periz et al., 2013). Rotavirus DLPs in cytoplasm produce capped, nonpolyadenylated, (+) ssRNA transcripts (from the negative strand of the genomic RNA) which are released from the particle through the class I channels (Fig. 1E and F). The transcripts serve either for translation of virus-encoded proteins (early in the replication cycle) or as templates for replication (late in the replication cycle) to become the dsRNA genomes of RV progeny (Silvestri et al., 2004). Particles of the Reoviridae family can produce mRNAs of a maximum of 12 different genomic segments (e.g. in the genus Coltivirus). For the Rotavirus (11 genomic RNA segments) and Orthoreovirus (10 genomic RNA segments) genera it is not clear whether the 1–2 ‘extra’ TCs are occupied by RNAs or are ‘empty’. RV DLPs are ‘transcriptionally active’ in the cellular cytoplasm and in vitro, where they produce large amounts (in mg range) of mRNAs, provided precursors and an energy source (ATP) are in sufficient supply (Cohen et al., 1979; Spencer and Arias, 1981; Lawton et al., 1997; Lu et al., 2008). A logarithmic increase in mRNA production at later stages

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Fig. 4. The rotavirus replication cycle. The rotavirus triple layered particles (TLPs) first attach to sialo-glycans (or histo-blood group antigens) on the host cell surface, followed by interactions with other cellular receptors, including integrins and Hsc70. Virus is then internalized by receptor-mediated endocytosis. Removal of the outer layer, triggered by the low calcium of the endosome, results in the release of transcriptionally active double-layered particles (DLPs) into the cytoplasm. The DLPs start rounds of mRNA transcription, and these mRNAs are used to translate viral proteins. Once enough viral proteins are made, the RNA genome is replicated and packaged into newly made DLPs in specialized structures called viroplasms, which interact with lipid droplets. The newly made DLPs bind to NSP4, which serves as an endoplasmic reticulum (ER) receptor, and bud into the ER. NSP4 also acts as a viroporin to release Ca2+ from intracellular stores. Transiently enveloped particles are seen in the ER. The transient membranes are removed as the outer capsid proteins VP4 and VP7 assemble, resulting in the maturation of the TLPs. The progeny virions are released through cell lysis. In polarized epithelial cells, particles are released by a non-classical vesicular transport mechanism. For further details see text. From Estes and Greenberg (2013). With permission of the authors and the publisher.

of infection (>4 h p.i.) indicates that the newly synthesized DLPs have become transcription active (secondary transcription) (StacyPhipps and Patton, 1987; Ayala-Breton et al., 2009). Rotavirus segment-specific mRNAs extruded from DLPs are then translated into the encoded proteins in the cytoplasm (see above and Table 2). 5.4. Viroplasm formation Rotavirus proteins and RNAs interact specifically in cytoplasmic inclusion bodies termed ‘viroplasms’. In order for viroplasms to form, the presence of two of the RV non-structural proteins, NSP2 and NSP5, is essential and sufficient, since co-expression of NSP2 and NSP5 in uninfected cells leads to the production of viroplasm-like structures (Fabbretti et al., 1999). Blockage of these two proteins by specific siRNAs, intrabodies or the use of NSP2- or NSP5-specific RV ts mutants at the non-permissive temperature prevent viroplasm formation, RV morphogenesis and the production of infectious RV progeny (Silvestri et al., 2004; Vascotto et al., 2004; Campagna et al., 2005). Recently, the conversion of ‘cytoplasmic into ‘viroplasm-associated by interaction with hypophosphorylated NSP5 has been observed, which is followed

by phosphorylation of both molecules (Criglar et al., 2014). Cytoplasmic NSP2 also forms complexes with VP1, VP2 and tubulin (Criglar et al., 2014), making tubulin a component of viroplasms (Martin et al., 2010), and induces microtubule depolymerization and stabilization by acetylation (Martin et al., 2010; Eichwald et al., 2012). Functional proteasomes and components of the autophagic pathway are essential for viroplasm formation and RV replication (Contin et al., 2011; López et al., 2011; Arnoldi et al., 2014). NSP2 forms octamers and has nucleoside triphosphatase, RNA helix destabilizing and nucleoside diphosphate kinase activities (Taraporewala et al., 1999; Taraporewala and Patton, 2001, 2004; Jiang et al., 2006). Grooves in the NSP2 octamer are binding sites for which NSP5 and ssRNAs compete, thus possibly regulating the balance between RV RNA translation and replication (Jiang et al., 2006). NSP5 is a serine- and threonine-rich protein which occurs in oligomeric forms and becomes hyperphosphorylated (Torres-Vega et al., 2000; Eichwald et al., 2004; Criglar et al., 2014). The exact biochemical functions of NSP5 remain to be determined (Campagna et al., 2007; Contin et al., 2010; Criglar et al., 2014). NSP4, a transmembrane glycoprotein, is mainly located in the endoplasmic reticulum (ER) and has multiple functions: (1) It


serves as an intracellular receptor for DLPs by interacting with VP6 (Taylor et al., 1996). (2) It releases Ca2+ from intracellular stores (Tian et al., 1994) by acting as a viroporin (Hyser et al., 2010, 2012), thus elevating the intracellular Ca2+ levels needed to stabilize the outer layer of TLPs. The viroporin function was found to be due to interaction of NSP4 with the cellular Ca2+ sensor molecule STIM1 (Hyser et al., 2013). (3) NSP4 forms caps on viroplasms and co-localizes with the autophagy protein LC3 (Berkova et al., 2006). The NSP4-triggered increase of intracellular (Ca2+ ) activates a kinase-dependent pathway, which leads to autophagy (Crawford et al., 2012; Crawford and Estes, 2013). (4) NSP4 alters plasma membrane permeability (Newton et al., 1997) and destabilizes intercellular junctions (Tian et al., 1996). (5) Most importantly, NSP4 was discovered to act as a viral enterotoxin (Ball et al., 1996): it is secreted early after RV infection, either as a peptide fragment or as a whole molecule (Zhang et al., 2000; Bugarcic and Taylor, 2006) and interacts with non-infected intestinal cells by integrin ␣1␤1 and ␣2␤1 receptors located at their basolateral plasma membranes (Seo et al., 2008). The activated integrin receptors stimulate intracellular reaction cascades which are correlated with intracellular Ca2+ release and diarrhea. NSP4 exists in 3 pools in the infected cell (Berkova et al., 2006): (1) localized in the ER as receptor for DLPs; (2) being secreted by the cell as a soluble component (Zhang et al., 2000; Bugarcic and Taylor, 2006); (3) forming ‘caps’ on viroplasms (Berkova et al., 2006). The RV NSP3 protein has been found to interact (at its N terminus) with the 3 terminus of viral (+) ssRNA and (at its C terminus) with the translation factor eIF4G (bound to the 5 terminus of the RNA), thus circularizing the viral RNA and functioning like the polyA binding protein (PABP) for cellular mRNAs (Piron et al., 1998, 1999; Vende et al., 2000; Groft and Burley, 2002). Since NSP3 can evict the PABP from cellular mRNAs, it prevents cellular mRNA translation very efficiently (Piron et al., 1998; Groft and Burley, 2002). Inhibition of NSP3 expression by siRNA, however, mainly suppresses its effect on cellular mRNA translation; viral RNA translation is not impaired (Montero et al., 2006). Cellular mRNA translation is inhibited by expressed NSP3 leading to the accumulation of PABP and of the cellular mRNAs themselves in the cell nucleus (Montero et al., 2006; Harb et al., 2008; Rubio et al., 2013). NSP3 also antagonizes part of the innate immune response (see below). NSP1 is the most variable of all RV proteins. It is likely involved in host range restriction (Broome et al., 1993; Feng et al., 2013). A key function of NSP1 is its ability to act as an antagonist of the innate immune response (see below). Viroplasms recruit cellular lipid droplets (LDs) which serve as energy store and transport vehicles in the cell (Cheung et al., 2010; Crawford et al., 2013). Analysis of the lipidome of viroplasm-LD complexes has confirmed their interaction (Gaunt et al., 2013b). Interference with LD homoeostasis and blockage of neutral fat breakdown decrease the number and size of viroplasms and the production and infectivity of viral progeny (Cheung et al., 2010; Crawford et al., 2013; Gaunt et al., 2013a). 5.5. RNA packaging, minus strand RNA synthesis and DLP formation Due to their persistence length of 1130 A˚ (Kapahnke et al., 1986), naked dsRNAs cannot be packaged into core shells. Rather, the 11 different (+) ssRNA segments are reassorted, interact with viral core proteins (see above) and are then packaged and replicated. It is assumed (Trask et al., 2012a) that primary replication complexes of VP1/VP3/ssRNA interact with a VP2 decamer (most likely involving NSP5 and NSP2) (Berois et al., 2003), leading to the formation of core particles. During this process, the negative charge of the RNA has to be neutralized by co-packaging of either divalent cations or

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spermidine, a cellular trivalent cationic compound (Gouet et al., 1999; Desselberger et al., 2013). The N-terminal domain of VP2 is essential for the encapsidation of VP1 (and the attached ssRNA) (Zeng et al., 1998; Boudreaux et al., 2013). Complex formation with VP2 is essential for the RdRp activity of VP1, with VP1/VP2 complexes constituting the minimal replicase particles in vitro (Zeng et al., 1996). The molecular details of the early morphogenesis of RV particles (core particle formation and RNA replication) are not well understood. In particular, it is unclear how the packaging of the correct set of 11 RNA segments into individual particles is controlled. Once formed, core particles are rapidly transcapsidated by VP6, leading to the synthesis of DLPs. The latter process has been studied in vitro (Trask and Dormitzer, 2006; Desselberger et al., 2013). 5.6. Virus particle (virion) maturation and release RV DLPs, upon leaving the viroplasms, bud through the endoplasmic reticulum (ER) for maturation. In this process, NSP4 serves as an intercellular receptor by interacting with VP6 (Taylor et al., 1996). Within the ER, nascent RV particles are transiently enveloped, but the envelope is lost when RV particles acquire the outer layer consisting of VP4 (60 trimers) and VP7 (260 trimers). (Estes and Greenberg, 2013). The origin, function and loss of the transitory envelope are not understood. However, it is likely that VP4 trimers, which form spikes, react with VP6 first and are then embedded in a continuous layer of VP7 trimers (Trask and Dormitzer, 2006) (Fig. 3). Transcapsidation experiments in vitro have shown that only by VP4 interacting with DLPs first, followed by interaction with VP7, full infectivity of TLPs can be restored (Trask and Dormitzer, 2006). Another pool of VP4 interacts with the cellular plasma membrane (González et al., 2000; Nejmeddine et al., 2000; Delmas et al., 2007). As described above, by interacting with DLPs NSP4 has a crucial function in the maturation process: blockage of NSP4 expression by siRNA leads to RV particle maturation defects (Silvestri et al., 2005), and RV RNA replication is also inhibited, possibly by the NSP4 fraction interacting with viroplasms (Berkova et al., 2006). RV TLPs are released from non-polarized cells (MA104) by lysis (McNulty et al., 1976), but from epithelial cells (e.g. Caco-2) by a kind of budding process that does not immediately kill the cell (Gardet et al., 2006). 5.7. Persistent RV infection In cell culture and in immunocompromised hosts (humans and other mammalian species) RV infection can become persistent (Estes and Graham, 1980; Chiarini et al., 1983; Riepenhoff-Talty et al., 1987; Mrukowicz et al., 1998). The detailed molecular mechanisms of this virus–host relationship are poorly understood at present. 5.8. Growth of RVs in stem cell organoids Following the achievement of differentiating human stem cell lines in vitro into intestinal cell-like cultures (human intestinal organoids) (Spence et al., 2011), it was shown that such cultures can be infected with RVs, both laboratory strains (SA11) and clinical isolates (Finkbeiner et al., 2012). The full potential of such cultures for studying RV replication is in the process of being explored. 5.9. Cellular proteins involved in RV replication As obligate cellular parasites viruses require cellular functions for their replication. Most viruses do not possess the machinery to translate proteins from their mRNAs and rely on the translation organelles and functions of the host cell. More recently, a much

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wider involvement of cellular compounds and functions in virus replication was uncovered. For RVs those are: cellular receptors and co-receptors for adsorption (section V.1), cellular endosomes for uncoating of TLPs to DLPs (section V.2), interaction of the ‘viral factory’ viroplasms with LDs (Section 5.4), interaction of NSP3 with components of the cellular translation machinery, - interaction of NSP4 with cellular membranes containing the intracellular Ca2+ stores (Section 5.4), - interaction of viral NS proteins with cellular structural proteins (actin, microfilaments), - interaction of NSP1 with various components of the cellular innate immune response (see below).

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6. RV genetics and reverse genetics RV functions have been explored by genetic studies with spontaneous mutants, but mainly by determining reassortment groups of ts mutant collections at the non-permissive temperature. Thus, for SA11 RV, 10 out of the 11 expected reassortment groups have been identified, and 9 of them assigned to individual genome segments (Criglar et al., 2011; Vende et al., 2013). Several reverse genetics (RG) systems for RVs have been described, but so far they all depend on the presence of a helper virus and require strong selection conditions. Thus, VP4 genes (native or chimeric) transcribed from a cDNA plasmid have been rescued into viable virus in the presence of neutralizing antibody directed against the VP4 of the helper virus (Komoto et al., 2006, 2011). A dual selection mechanism (use of ts mutants of the helper virus at the non-permissive temperature, and of siRNA directed against the helper virus gene of interest for engineered reassortment) has been applied to drive single gene RG (Trask et al., 2010b) (Fig. 5). Using this technique, it has been possible to engineer partial gene duplications and heterologous cDNA sequences (encoding FLAG, a hepatitis C virus E2 epitope and an IRES sequence) into the 3 UTR of the NSP2 gene of the SA11 RV (Navarro et al., 2013), thus opening the potential of creating a recombinant RV that can act as a viral vector. In addition, rearranged genome segments transcribed from cDNA were engineered into helper virus, possibly based on the preferential packaging of rearranged genes (Troupin et al., 2010). Attempts to establish a helper virus-free, fully tractable and universally applicable RG system for RV have so far failed, in contrast to successful, RNA- or plasmid-only based RG systems for orthoreovirus (Kobayashi et al., 2007) and orbivirus (Boyce et al., 2008). Following the lead of Boyce et al. (Boyce et al., 2008), who rescued infectious bluetongue virus after co-transfection of the 10 full-length (+) ssRNA transcripts, Richards et al. (Richards et al., 2013) were unable to rescue viable RVs. The main impediment seemed to be a lack of translation of transfected full-length RV RNAs or cDNAs (with the RV insert downstream from a T7 promoter, in the presence of a fowlpox virus-T7 recombinant) and a strong cytotoxic effect of transfected RNAs. O’Neill et al. (2013), Wentzel et al. (2013) and Wentzel (2014) observed translation of several transfected full-length RNA segments, possibly by using different cell lines, but also recorded a high degree of cytotoxicity which they considered to be caused by an extremely high stimulation of the innate immune response. Despite progress in obtaining translation of transfected RV RNAs, this research group could not rescue infectious RV progeny from RNA co-transfections alone, either. Taken together, work on plasmid-only or transcriptonly based RG systems for RVs has so far only succeeded in helping to identify procedural bottlenecks which have to be overcome. Despite the lack of a nucleic acid-only based RG system, it has been possible to incorporate in vivo-biotinylated VP6 into

infectious RV particles (De Lorenzo et al., 2012); the procedure should be useful for labelling and functional exploration of other RV structural proteins.

7. Molecular pathogenesis and pathophysiology RVs mainly infect mature enterocytes at the top of the villi of the small intestine of mammalian species, where vacuolization and epithelial loss can be observed, followed by crypt hyperplasia. Although extraintestinal spread of RV occurs frequently as evidenced by detection of RV dsRNA, RV antigen, and (occasionally) infectious RV in serum and other host body sites (Blutt et al., 2003; Blutt and Conner, 2007), the significance of these observations for pathologic findings in normal (immunocompetent) hosts is controversial (Fenaux et al., 2006; Ramig, 2007; Ramani et al., 2010). By contrast, in the immunocompromised hosts, RV can replicate in the liver, the biliary system and the pancreas and be associated with biliary atresia and pancreatitis (Gilger et al., 1992; Feng et al., 2008). A striking finding (in animal models) is the onset of diarrhea at an early time point when histopathological changes of the small intestine are absent (Ward et al., 1996), likely due to early action of NSP4 (Estes and Morris, 1999; Estes and Greenberg, 2013) which forms oligomers of different size (Bowman et al., 2000; Chacko et al., 2011). The pathogenesis of RV disease is multifactorial, depending on the age of host, homology/heterology of virus-host interaction, and particular viral gene products (VP3, VP4, VP7, NSP2, NSP3, NSP4; Broome et al., 1993; Ball et al., 1996; Burke and Desselberger, 1996; Estes and Atmar, 2003; Ramig, 2004; Greenberg and Estes, 2009). Using simian (RRV)-mouse (EW) RV reassortants, Feng et al. (2013) concluded that the genes encoding the mouse VP4 and NSP1 were determinants for efficient replication in suckling mice. Factors of the disease mechanism are: maladsorption following destruction of epithelium (Estes and Atmar, 2003), villus ischemia (Osborne et al., 1991), the action of NSP4, a viral enterotoxin (Ball et al., 1996; Greenberg and Estes, 2009; Chattopadhyay et al., 2013), and activation of the enteric nervous system (Lundgren et al., 2000). Recently, the pathogenesis of the symptom vomiting has also been elucidated by observations that RV can infect the enterochromaffine cells in the gut and stimulate the production of 5-hydroxytryptamin (serotonin) which in turn activates vagal afferent nerves and stimulates brain stem structures controlling vomiting (Hagbom et al., 2011, 2012). Biliary atresia (BA) can be induced in newborn mice by RV infection, leading to obstructive cholangiopathy, the severity of which depends on the host’s age at the time of infection and also on the presence and potency of innate immune responses (Mohanty et al., 2013). In this model, newborn mice can be protected from BA by passive immunization, achieved either by immunizing the dams with RV-like particles or by injecting hyperimmune serum into pubs prior to infection (Hertel et al., 2013). A relatively novel area of the exploration of gut diseases is the metagenomic analysis of viruses and other microbes found in the gut (Finkbeiner et al., 2008; Minot et al., 2012, 2013) and the correlation of their presence with disease. Using this approach, genomes of viruses can be discovered the particles of which have not been previously observed nor isolated by cell culture. So far, mainly DNA viruses, many of them bacteriophages, were analyzed. Protocols to discover both DNA and RNA viruses in fecal specimens have been developed (Sachsenroder et al., 2012), and applications to the analysis of the full human gut virome have started (Kapusinszky et al., 2012; Phan et al., 2012; Ryan, 2013). It will be important to know whether and to what extent the human gut microbiome, including the virome, may influence the pathogenesis of chronic gut diseases (such as Crohn’s disease and colitis ulcerosa) (Virgin, 2014). Using the gnotobiotic piglet model of human RV infection and disease, it


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Fig. 5. Reverse genetics of a gene 8 recombinant rotavirus using a dual selection system. (A) Diagram of the recombinant gene 8 cDNA. A minimal T7 RNA polymerase promoter (T7P) initiates transcription of an SA11 gene 8 (+) RNA with an authentic 5 end. The g8D RNAi target site was engineered to prevent targeting of the recombinant mRNA. Cleavage by a modified HDV ribozyme (Rbzm) generates the authentic 3 end. (B) Diagram of the reverse genetics method. COS-7 cells are infected with rDIs-T7pol (VV-T7) (i) and transfected with the recombinant gene 8 plasmid (pBS-SA11g8R) (ii) before infection with the tsE helper virus at 30 ◦ C (iii). The recovered virus stock contains both tsE helper virus and the tsE/SA11g8R virus. Passage at 39 ◦ C in MA104-g8D cells (iv) allows rapid isolation of the tsE/SA11g8R virus (v). From Trask et al. (2010b). With permission of the authors and the publisher.

was observed that properly dosed probiotic lactic acid bacteria can enhance the Th1 and Th2 cytokine responses and partially prevent RV-induced injuries to the gut epithelium (Azevedo et al., 2012; Liu et al., 2013). 8. Immune responses 8.1. RV-specific humoral and cell-mediated immune responses Upon RV infection, acquired immune responses are elicited, both from B cells producing antibodies directed against virusspecific proteins, and from T cells recognizing T cell-specific RV epitopes on the surface of infected cells in complexes with MHC classes I and II antigens. Many antibodies directed against VP7 and VP4 on the surface of RV particles are neutralizing (NAb) in vitro and protective in vivo, as demonstrated by passive transfer in mice and gnotobiotic piglets as animal models (Offit, 1994; Feng et al., 1997; Franco and Greenberg, 1997; Yuan and Saif, 2002; Jiang et al., 2002). Passive transfer of RV-specific CD8+ T cells has also shown to be protective (Offit, 1994). Transplacentally acquired RV-specific antibodies likely protect newborns from infection (Ray et al., 2007) and interfere with immune responses to RV vaccination (Johansson et al., 2008; Appaiahgari et al., 2014). The availability of various knockout mutant mice has permitted researchers to dissect the relative contributions of humoral and cellular immune responses to protection: while RV-specific T cells help eliminate RV after primary infection, it is the RV-primed memory B cells which provide more long-term protection (Franco et al., 2006). Humoral antibodies boosted after repeated infection are directed against both serotypespecific and cross-reactive epitopes on VP4 and VP7 molecules, thus

also providing heterotypic protection (Franco et al., 2006). Plasmacytoid dendritic cells were found to be necessary and sufficient to induce B cell activation after RV infection in mice (in vivo) and in human cells (in vitro) (Deal et al., 2013). Human RV-specific CD4+ T cells circulating in the blood express the intestinal homing receptor ␣4␤7 (Gonzalez et al., 2003; Weitkamp et al., 2005; Parra et al., 2014). However, protection from RV infection is not entirely correlated with the concentration of VP4- and VP7-specific NAbs. Upon natural infection by or vaccination with RV, infants and young children develop antibodies against other structural (VP6, VP2) and nonstructural (NSP4) proteins. VP6-specific antibodies do not neutralize in vitro, but were shown to be protective in vivo (Burns et al., 1996), when VP6-specific antibodies of the IgA class were applied. It was suggested and has recently been proven that VP6specific IgA antibodies are taken up (‘transcytosed’) by epithelial gut cells through J protein receptors at the basolateral membrane and form complexes with new DLPs released from viroplasm, thus preventing their maturation to TLPs (‘intracellular neutralization’) (Desselberger, 1998; Schwartz-Cornil et al., 2002; Feng et al., 2002; Corthésy et al., 2006; Aiyegbo et al., 2013; Sapparapu et al., 2013). However, it has also been shown that mice and patients who are IgA-deficient eliminate RV after infection, probably due to a compensatory IgG protective immunity (O’Neal et al., 2000). Passive transfer of NSP4 antibody has produced some protection in mice (Hou et al., 2008), but actively acquired NSP4 antibody does not protect gnotobiotic piglets from a challenge with RVA (Yuan et al., 2004). Prospective studies have shown that, after 1 or 2 natural RV infections, children appear to be highly protected against severe disease following infection by various, also heterotypic, RVs (Velázquez et al., 1996).

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thus delaying cell death during the early stages of RV replication (Bhowmick et al., 2013). Combined flow cytometry and single cell multiplex RT-PCR allowed the dissection of the IIRs of RV-infected and bystander intestinal epithelial cells in mice infected with a homologous RV; heterogeneity of induction and subversion of early IIRs was discovered (Sen et al., 2012). Rotavirus infection causes more severe disease in mice deficient in IFN signaling (Vancott et al., 2003; Feng et al., 2008, 2011). Interferon production is a dominant IIR in RV-infected pigs (González et al., 2010) and humans (Wang et al., 2007). Human plasmacytoid dendritic cells do not replicate RV, but initiate a brisk IFN response that may stimulate B cell responses (Deal et al., 2013). The virus-encoded nonstructural protein NSP4 secreted from RV-infected cells was shown to induce the synthesis of proinflammatory cytokines from murine macrophages (Ge et al., 2013). Human RV-specific CD4+ and CD8+ T cells and B cells express the gut homing receptor ␣4␤7 (Rojas et al., 2003, 2008). Thus, RV components interact by various mechanisms with molecules and pathways of the cellular IIR. 8.3. Correlates of protection Fig. 6. Modulation of the host innate immune system by RV infection. After entry into cells, actively transcribing RV is recognized by receptors RIG-I and MDA-5 which activate the transcription factors IRF3 and NF-kB; in turn those migrate to the nucleus and stimulate interferon (IFN) and IFN stimulatory genes (ISG). The viral NSP1 interacts with IRF3 and other cellular proteins (NF-␬B a.o.), which are then degraded. Interferon is secreted and, by binding to IFN receptors, activates transcription factors STAT-1, STAT-2, and IRF9 which in turn further stimulate IFN production and establish an ‘antiviral state’. For further details see text. From Angel et al. (2012). With permission of the authors and the publisher.

8.2. Innate immune responses RV infection immediately triggers various mechanisms of innate immune responses (IIR), which occur earlier than acquired RVspecific immune responses, at least after primary infection (Angel et al., 2012) (Fig. 6). Many IIRs appear to be RV strain-specific and also cell type-specific (Feng et al., 2009). At present it is not fully clear to what extent the IIRs after RV infection modify disease. It has been shown that the RV NSP1 is able to interact with the following cellular proteins: interferon (IFN) regulatory factors (IRF) 3 (Graff et al., 2002, 2007; Barro and Patton, 2005; Sen et al., 2011), ␤-transducin repeat containing protein (␤-TrCP0) (Graff et al., 2009; Qin et al., 2011), melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) (Broquet et al., 2011; Sen et al., 2011; Nandi et al., 2014), the tumor suppressor protein p53 (Bhowmick et al., 2013) and the TNF receptor associated factor 2 (TRAF2) (Bagchi et al., 2013), leading to their proteasomal degradation and thus preventing or down-regulating the early triggering of an IFN response. RV NSP1 also induces downregulation of IRF5 and IRF7 (Barro and Patton, 2007) and targets retinoic acid-inducible gene I (RIG-I) (Broquet et al., 2011) which, however, is downregulated by a proteasome-independent pathway (Qin et al., 2011). NSP1 interacts with IRF3 by binding to the dimerisation domain of the protein (Arnold et al., 2013a, 2013b). Rotavirus infection also inhibits STAT1 phosphorylation and STAT1/STAT2 translocation to the nucleus and thereby the establishment of an IFN-induced ‘antiviral state’ (Holloway et al., 2009, 2014; Holloway and Coulson, 2013; Sen et al., 2014). In addition, the C-terminal domain of the RV capping enzyme, VP3, has recently been shown to contain a phosphodiesterase which cleaves 2 ,5 -oligoadenylate, thereby preventing activation of RNase L and blocking a potent IIR host cell response (Zhang et al., 2013). RIG-I was found to act as a cytoplasmic sensor for ssRNAs extruded from transfected DLPs (Uzri and Greenberg, 2013). NSP1 targets the pro-apoptotic cellular protein p53, leading to its proteasomal degradation and

In general, there is a correlation between the presence of certain levels of humoral RV-specific antibody and protection (e.g. RV-specific copro IgA of >1:80; RV-specific serum IgA of >1:200; RV-specific serum IgG of >1:800) (Matsui et al., 1989a, 1989b; Offit, 1994). The significance of RV-specific serum IgA antibody levels as correlates of protection has been claimed (Blutt et al., 2012; Patel et al., 2013; Cheuvart et al., 2013; Blutt and Conner, 2013), but this remains controversial (Angel et al., 2012). However, since there is a lack of correlation between relatively low Nab titers after natural RV infection or vaccination and high levels of protective immunity achieved, additional factors of importance for protection are likely (Desselberger and Huppertz, 2011). Interestingly, both currently licensed RV vaccines are highly efficacious and effective in countries of temperate climate (see below), although the correlates of protection are incompletely defined (Franco et al., 2006; Angel et al., 2007; Desselberger and Huppertz, 2011). 9. Clinical symptoms and differential diagnosis, laboratory diagnosis and treatment RV infections can remain asymptomatic (see below) or lead to acute gastroenteritis (AGE) with mild to severe diarrhea, vomiting and various degrees of dehydration. The body temperature is often, but only moderately, elevated. Vomiting precedes diarrhea and lasts for a shorter time period (3 vs 5 days). A massive electrolyte imbalance leads to often severe dehydration. Death from RV disease is mainly due to severe dehydration and cardiovascular failure. Whereas death from RV-associated disease is rare in developed and semi-developed countries, mainly due to the timely availability of rehydration measures, it is a frequent outcome in countries of subSaharan Africa and southeast Asia (Tate et al., 2012). Upon natural infection or (inadvertent) vaccination, children with severe combined immunodeficiency (SCID) can develop a chronic RV infection and disease (Desselberger, 1996; Patel et al., 2010). In chronically infected children, RVs with ‘unusual’ RNA migration patterns emerge, due to genome rearrangements (Desselberger, 1996). The diagnosis of RV infection is by electron microscopy or ELISA (Brandt et al., 1981), and more recently by RT-PCR (Gouvea et al., 1990; Gentsch et al., 1992; Gómara et al., 2000; Iturriza-Gómara et al., 2004). Of those procedures, ELISA is applied most frequently in the routine diagnostic laboratory (due to the ease of use and speed of obtaining a result). However, RT-PCR, which is highly sensitive and specific (Richardson et al., 1998) and also suitable


for genotyping (Fischer and Gentsch, 2004; Iturriza-Gómara et al., 2004), has become the ‘gold standard’ of diagnostic discovery. Due to continuous genomic drift by point mutations (see below) the primers used for genotyping have to be changed periodically (Iturriza-Gómara et al., 2000, 2004; Simmonds et al., 2008). For the differential diagnosis of disease causes, the main viruses to be considered are human caliciviruses (norovirus, sapovirus), astroviruses and (less frequently) enteric adenoviruses and Aichi virus. Many of those can be searched for by multiplex RT-PCR assay (e.g., Liu et al., 2011). Other viruses infecting the gut and remaining mostly asymptomatic are different enteroviruses and, in the immunocompromised, picobirnaviruses, cytomegalovirus and HIV (Desselberger and Gray, 2013). In an internally controlled study, the genomes of G10P[11] RVs isolated from 20 asymptomatic and 19 symptomatic neonates in Vellore, India, in 2003/2004, were determined by dideoxynucleotide (Sanger) and next generation sequencing and found to be virtually identical. Most importantly, no nt or amino acid (aa) differences were found which were correlated with asymptomatic or symptomatic infection (Libonati et al., 2014). It was concluded that other, poorly defined factors would determine the appearance of clinical symptoms. The treatment consists mainly of rehydration (orally or intravenously) (American Academy of Pediatrics, 1996; Desselberger, 1999; Atia and Buchman, 2009; Guarino et al., 2012). Various compositions of oral rehydration salts (ORS) have been devised (CHOICE Study Group, 2001; Atia and Buchman, 2009), and rice-based solutions are also successfully applied (Pizarro et al., 1991). The use of loperamide, anticholinergic drugs and adsorbents is not recommended (American Academy of Pediatrics, 1996). Racecadotril, an encephalinase inhibitor suppressing the enteric nervous system activated after RV infection (Lundgren et al., 2000; Salazar-Lindo et al., 2000), granisetron, a serotonin receptor antagonist, and vasoactive intestinal peptide antagonists (Kordasti et al., 2004) were also found to be beneficial in decreasing the severity of acute diarrhea. Oral solutions containing high concentrations of RV antibody were found to be beneficial therapeutically, as well as a preventative measure (Saulsbury et al., 1980; Ebina, 1996). Recently, llama-derived single-chain antibody fragments specific for RV VP6 were shown to be broadly cross-reactive in vitro and to decrease virus shedding in infected mice (van der Vaart et al., 2006; Garaicoechea et al., 2008; Aladin et al., 2012). This antibody was also efficacious in decreasing RV replication in gnotobiotic piglets (Vega et al., 2013) and had a moderately beneficial effect on RV disease in humans (Sarker et al., 2013; Kang, 2013; Tokuhara et al., 2013). The antibody fragment that is of only 15 kDa size (Joosten et al., 2003) remained active after long-term (>1 year) storage and was resistant to heating at 94 ◦ C for 90 min (Tokuhara et al., 2013). The llama antibody fragment was expressed at high levels in transgenic rice (MucoRice-ARP1); in pilot studies, it suppressed the RV load in both immunocompetent and immunodeficient mice and also conferred protection from RV infection (Tokuhara et al., 2013). This and similar antibody preparations have a realistic potential for therapeutic and prophylactic use in humans (Kang, 2013). Other therapeutic approaches consist of the use of the broad spectrum antiviral nitazoxanide (Rossignol et al., 2006; Teran et al., 2009; La Frazia et al., 2013) and – supported by work with gnotobiotic piglets (Azevedo et al., 2012; Liu et al., 2013) – of probiotics (Fang et al., 2009; Grandy et al., 2010; Corrêa et al., 2011; Ciccarelli et al., 2013).

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require treatment in primary care centers or hospitals (Malek et al., 2006; Parashar et al., 2006; Liu et al., 2012). In the US before vaccination, RVs caused 3 million disease episodes per annum, requiring 500,000 visits to a medical doctor and 60,000 hospitalisations, but leading to only 20–40 deaths (Fischer et al., 2007; Esposito et al., 2011). Similar numbers were found to be valid in Europe, except that the number of deaths was about 200 (Soriano-Gabarró et al., 2006; Van Damme et al., 2007). In sub-Saharan African and in some south Asian countries, RV disease is associated with a high mortality (Parashar et al., 2009; Tate et al., 2012). Despite the large genomic and antigenic diversity of RVs (see above), globally only a small number of RV types have prevailed in humans during the past 3 decades: RVs of types G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8], and more recently G9P1A[8] and G12P1A[8] have co-circulated at high frequency, contributing 80–90% of all RV infections in North America, Europe and Australia (Gentsch et al., 2005; Santos and Hoshino, 2005; Iturriza-Gómara et al., 2011). By contrast, in African, Asian and South American countries, other genotypes such as G5, G6 and G8 are more prevalent (Todd et al., 2010; Mwenda et al., 2010; da Silva et al., 2011; Kang et al., 2013; Luchs and Timenetsky, 2014). Regarding the overall prevalence estimates of particular RVA genotypes, the problem of sampling bias should not be underrated. With the advent of RV genotyping by RT-PCR (Gouvea et al., 1990; Gentsch et al., 1992), detailed studies of the molecular epidemiology of RVs have become possible. Genotyping has an enormous value for assessing the evolution and epidemiological pathways of RVs in humans, mammals and birds (Matthijnssens et al., 2011b; Matthijnssens and Van Ranst, 2012). However, the importance of RV typing for success of vaccination may have been overestimated. Among other facts, the broad heterotypic efficacy and effectiveness of a monovalent RV vaccine (Rotarix® ) calls this ‘dogma’ into question (see below). Outbreak of AGE caused by species B RVs have been observed in China (Hung et al., 1983, 1984), and individual cases of species B RV infections have been detected in Bangladesh and India (Kelkar and Zade, 2004; Lahon et al., 2013). RVC infections are mostly asymptomatic or clinically mild, but can cause diarrhea in adults (Nilsson et al., 2000). Infections with RVD, RVF and RVG are mainly discovered in birds. Infections with RVH were detected in piglets (Wakuda et al., 2011; Molinari et al., 2014; Marthaler et al., 2014) and in humans (Jiang et al., 2008b). RVs are very contagious for the following reasons: (1) a low number of virus particles at a low ratio of (number of virus particles/infectious units) suffices to productively infect a susceptible individual (Ward et al., 1986; Graham et al., 1987); (2) rotavirus particles are shed in large numbers in feces (up to 1011 particles/ml) during the acute stage of the infection (Ward et al., 1984) or for longer periods by infected immunocompromised hosts (Patel et al., 2010); and (3) RV particles are very resistant to environmental conditions (temperature, pH, etc.; Estes et al., 1979; Keswick et al., 1983). The transmission is usually fecal–oral, the incubation period is short (1–2 days). Attendance of day child care centers is a risk factor to acquire a symptomatic RV infection (Pickering et al., 1988; Dennehy et al., 2006). Small RV epidemics among the elderly (in old people’s homes, geriatric wards, etc.) have been observed (Hrdy, 1987). Nosocomial RV infections are frequent and difficult to eliminate (Gleizes et al., 2006). The evolution of RVs has been elucidated by widespread genome-wide RT-PCR genotyping supported by cDNA sequencing (Matthijnssens and Van Ranst, 2012). Several mechanisms were identified (Iturriza-Gomara et al., 2003):

10. Molecular epidemiology and mechanisms of evolution Rotaviruses cause 5–10% of all cases of AGE in infants and young children of 90%) in developed countries (Patel and Parashar, 2009; Boom et al., 2010; Vesikari et al., 2010a, 2010b; Anderson et al., 2011; Buttery et al., 2011b; Giaquinto et al., 2011; Quintanar-Solares et al., 2011; Tate et al., 2011a, 2011b, 2013; Tregnaghi et al., 2011; Desai et al., 2012; Payne ˜ et al., 2013; Rha et al., et al., 2013a; Cortese et al., 2013; Gastanaduy 2014; Leshem et al., 2014), significantly decreasing RV-associated AGE necessitating visits to medical practitioners and hospitalization, and in some instances also decreasing mortality from the ˜ et al., 2013). There condition (Richardson et al., 2010; Gastanaduy is no clear difference in efficacy between the two vaccines. As a surprising but welcome side effect, ‘herd immunity’ in non-vaccinated children was observed (Yi and Anderson, 2013; Anderson et al., 2013; Dennehy, 2013; Leshem et al., 2014). There remains a very small vaccine-attributable risk for IS for both vaccines, at approximately 1:50,000 or less, which, however, is exceeded by the benefits of the vaccines (Buttery et al., 2011a; Patel et al., 2011; Greenberg, 2011; Haber et al., 2013; Carlin et al., 2013; Parashar and Orenstein, 2013; Glass and Parashar, 2014; Weintraub et al., 2014; Yih et al., 2014). In contrast to the high effectiveness of RV vaccination in developed countries, in some middle and low income countries, such as Mexico, Nicaragua, Malawi, South Africa, Kenya, Mali, Bangladesh and Vietnam, the efficacy of the vaccine was found to be substantially (20–30%) lower (Armah et al., 2010; Madhi et al., 2010; Zaman et al., 2010) for reasons that are at present not fully understood (Lopman et al., 2012; Glass et al., 2014). One factor may be vitamin A deficiency, as experimentally explored in gnotobiotic piglets (Vlasova et al., 2013; Chattha et al., 2013; Kandasamy et al., 2014).


Since the mortality from RV-associated AGE is highest in the low income countries (Tate et al., 2012), WHO recommends since 2009 that RV vaccination should be carried out in all countries, accepting the relatively lower vaccine effectiveness in low income countries (SAGE, 2009; WHO, 2013). The finding of porcine circovirus type 1 and 2 DNA, detected in the two licensed vaccines by metagenomic techniques, was initially a shock, but then investigated and not found to be harmful for humans, and the use of the vaccines continues (Victoria et al., 2010; McClenahan et al., 2011; Dubin et al., 2013; Esona et al., 2014). Although the monovalent RV1 vaccine is highly effective against heterotypic G2P[4] RVs, the proportion of G2P[4]-associated AGE was higher in vaccinated than in unvaccinated hospitalized children (Matthijnssens et al., 2014). Others did not find a significant, vaccine-attributable change in the prevalence of particular RV genotypes (Dennehy, 2013; Hemming and Vesikari, 2013a; PeláezCarvajal et al., 2014; Donato et al., 2014). To obtain more clarity about this problem, the post-marketing surveillance of RV strains and genotype prevalence is continuing. Since both RV vaccines contain live attenuated virus, it is not surprising that RV vaccine strain reassortants (between both RV vaccine strains and vaccine and wt strains which had arisen during natural co-circulation) were discovered (Bucardo et al., 2012; Boom et al., 2012; Hemming and Vesikari, 2013b). Another live attenuated RV, the human-bovine natural reassortant 116E (G9P8[11], originally isolated from asymptomatically infected newborns in India), has been tested in a phase III clinical trial in India, and an efficacy against severe RV disease of 56% was recorded (Bhandari et al., 2014) which is comparable with that of the R1 and R5 RV vaccines in middle and low income countries (see above). Due to the high effectiveness of RV vaccine programs, the predominant cause of hospitalization of children with AGE are now norovirus infections (Koo et al., 2013; Payne et al., 2013b; Hemming et al., 2013; Bucardo et al., 2014). As alternatives to live attenuated vaccines, inactivated virus particles (Jiang et al., 2008a, 2013; Wang et al., 2010), virus-like particles (VLPs) expressed from baculovirus recombinant-infected insect cells (Crawford et al., 1994; Conner et al., 1996; O’Neal et al., 1997; Ciarlet et al., 1998; Crawford et al., 1999; Azevedo et al., 2013) or yeast (Rodríguez-Limas et al., 2014), and DNA-based vaccines (Herrmann et al., 1996) are being explored. Rotavirus VP6 nanotubules, also in combination with norovirus VLPs, are promoted as candidates for novel RV vaccines (Tamminen et al., 2013; Lappalainen et al., 2013; Rodríguez et al., 2013; Pastor et al., 2014). Experimentally, VP6-specific single chain antibodies (of only 15 kDa MW) produced in llamas have been shown to have some cross-protective effect in vitro (Garaicoechea et al., 2008) and in vivo (Garaicoechea et al., 2008; Vega et al., 2013). 12. Perspectives of future basic and translational research Major progress in basic RV research will depend on the establishment of a tractable, helper virus-independent, plasmid (cDNA)or RNA-only based reverse genetics system for RVs. Based on the data reviewed, the following topics will require or are likely to attract research attention in the future: - the 3D structure of the dsRNA segments packaged in the RV core and their potential interaction with the core shell VP2; - the different receptor specificities of different RV strains; - details of the interaction of viroplasms with lipid droplets; - the mechanisms controlling assortment/reassortment of 11 RNA segments in replication complexes (i.e. RNA/RNA interactions); - the mechanisms controlling RV RNA packaging;

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- the formation and loss of temporary enveloped RV particles in the ER during the viral maturation process; - the potential of intestinal organoid cell cultures for the study of RV replication; - the factors (mutations) determining RV pathogenicity and virulence; - the molecular basis for host range and organ/cell specific viral replication; - the significance of the different arms of the immune response in establishing protection against RV disease; - the full identification of the correlates of protection against RV disease; - the factors determining RV spread; - the reasons for decreased efficacy of RV vaccines in reservestrapped settings and areas of low socioeconomic conditions; - the further evolution of human RVs under the condition of universal mass vaccination against RV disease; - the development of alternative (non-live attenuated) RV vaccine candidates; - the development of RV antivirals. Acknowledgements The author’s work on rotaviruses has been made possible over the years by various grants, mainly from The Wellcome Trust and the MRC, both U.K., and by institutional support from the CNRS, Gif-sur-Yvette, France (co-investigator: Dr J. Cohen), the ICGEB, Trieste, Italy (co-investigator: Dr O. Burrone) and the Department of Medicine, University of Cambridge, Cambridge, U.K. (co-investigator: Professor A Lever). The chapter on Rotaviruses by MK Estes and HB Greenberg in Fields Virology, 6th edition, 2013, has provided excellent guidance for this review. References Adams, W.R., Kraft, L.M., 1963. Epizootic diarrhea of infant mice: identification of the etiologic agent. Science 141 (July (3578)), 359–360. Aiyegbo, M.S., Sapparapu, G., Spiller, B.W., Eli, I.M., Williams, D.R., Kim, R., Lee, D.E., Liu, T., Li, S., Woods Jr., V.L., Nannemann, D.P., Meiler, J., Stewart, P.L., Crowe Jr., J.E., 2013. Human rotavirus VP6-specific antibodies mediate intracellular neutralization by binding to a quaternary structure in the transcriptional pore. PLoS ONE 8 (May (5)), e61101. Aladin, F., Einerhand, A.W., Bouma, J., Bezemer, S., Hermans, P., Wolvers, D., Bellamy, K., Frenken, L.G., Gray, J., Iturriza-Gómara, M., 2012. In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llamaderived antibody fragments. PLoS ONE 7 (3), e32949. American Academy of Pediatrics, 1996. Practice parameter: the management of acute gastroenteritis in young children. Provisional Committee on Quality Improvement, Subcommittee on Acute Gastroenteritis. Pediatrics 97, 424–435. Anderson, E.J., Rupp, A., Shulman, S.T., Wang, D., Zheng, X., Noskin, G.A., 2011. Impact of rotavirus vaccination on hospital-acquired rotavirus gastroenteritis in children. Pediatrics 127 (February (2)), e264–e270. Anderson, E.J., Shippee, D.B., Weinrobe, M.H., Davila, M.D., Katz, B.Z., Reddy, S., Cuyugan, M.G., Lee, S.Y., Simons, Y.M., Yogev, R., Noskin, G.A., 2013. Indirect protection of adults from rotavirus by pediatric rotavirus vaccination. Clin. Infect. Dis. 56 (March (6)), 755–760. Angel, J., Franco, M.A., Greenberg, H.B., 2007. Rotavirus vaccines: recent developments and future considerations. Nat. Rev. Microbiol. 5 (July (7)), 529–539. Angel, J., Franco, M.A., Greenberg, H.B., 2012. Rotavirus immune responses and correlates of protection. Curr. Opin. Virol. 2 (August (4)), 419–425. Appaiahgari, M.B., Glass, R., Singh, S., Taneja, S., Rongsen-Chandola, T., Bhandari, N., Mishra, S., Vrati, S., 2014. Transplacental rotavirus IgG interferes with immune response to live oral rotavirus vaccine ORV-116E in Indian infants. Vaccine 32 (February (6)), 651–656. Arista, S., Giammanco, G.M., De Grazia, S., Colomba, C., Martella, V., Cascio, A., Iturriza-Gòmara, M., 2005. G2 rotavirus infections in an infantile population of the South of Italy: variability of viral strains over time. J. Med. Virol. 77 (December (4)), 587–594. Armah, G.E., Sow, S.O., Breiman, R.F., Dallas, M.J., Tapia, M.D., Feikin, D.R., Binka, F.N., Steele, A.D., Laserson, K.F., Ansah, N.A., Levine, M.M., Lewis, K., Coia, M.L., Attah-Poku, M., Ojwando, J., Rivers, S.B., Victor, J.C., Nyambane, G., Hodgson, A., Schödel, F., Ciarlet, M., Neuzil, K.M., 2010. Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial. Lancet 376 (August (9741)), 606–614.

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Rotaviruses: is their surveillance needed?

Different serotypes of human rotaviruses.

Rotaviruses and the respiratory tract.

Distinguishing the genotype 1 genes and proteins of human Wa-like rotaviruses vs. porcine rotaviruses.

Production and characterization of monoclonal antibodies to porcine group C rotaviruses cross-reactive with group A rotaviruses.

Editorial: Rotaviruses of man and animals.

Transmission of human rotaviruses to gnotobiotic piglets.

Routine diagnosis of human rotaviruses in stools.

Reassortant human group C rotaviruses in Hungary.

Antigenic relationship between human and simian rotaviruses.

In-vitro detection of human rotaviruses.

Porcine Rotaviruses: Epidemiology, Immune Responses and Control Strategies.

Inactivation of human and simian rotaviruses by chlorine dioxide.

Experimental Adaptation of Rotaviruses to Tumor Cell Lines.

Entirely plasmid-based reverse genetics system for rotaviruses.

Evidence for two serotype G3 subtypes among equine rotaviruses.

Reactivities of serotyping monoclonal antibodies with culture-adapted human rotaviruses.

Evidence of serologic diversity within group C rotaviruses.

Global occurrence and emission of rotaviruses to surface waters.

Rotaviruses.

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