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. 2022 Dec 28;324:199032. doi: 10.1016/j.virusres.2022.199032

14th International dsRNA Virus Symposium, Banff, Alberta, Canada, 10-14 October 2022

Ulrich Desselberger 1
PMCID: PMC10242350  PMID: 36584760

Highlights

  • •

    Keynote address by Nihal Altan-Bonnet, NIH on ‘Non-lytic release of enteric viruses in cloaked vesicles’.

  • •

    Jean Cohen Lecture by Polly Roy, London School of Hygiene and Tropical Medicine, on ‘The replication cycle of bluetongue virus reveals its secrets, and those it shares with rotaviruses’.

  • •

    Genomic diversity of dsRNA viruses.

  • •

    Structure-function studies of dsRNA viruses (transcriptomics of pathogenesis, reverse genetics applications, biomolecular condensates during virus replication, viral FAST proteins, applications of mammalian intestinal organoids).

  • •

    Rotavirus vaccine development and potential use as vectored vaccine candidate.

Keywords: dsRNA viruses, Reovirus, Rotavirus, Mycovirus, Toti-like virus, FAST proteins, Viruscell receptor interaction, Biomolecular condensates, Liquid-liquid phase separation, Spatiotemporal transcriptomics, Reverse genetics, Next generation rotavirus vaccines, Rotavirus recombinants as vectored vaccine candidates

Abstract

This triennial International dsRNA Virus Symposium covered original data which have accrued during the most recent five years. In detail, the genomic diversity of these viruses continued to be explored; various structure-function studies were carried out using reverse genetics and biophysical techniques; intestinal organoids proved to be very suitable for special pathogenesis studies; and the potential of next generation rotavirus vaccines including use of rotavirus recombinants as vectored vaccine candidates was explored.

'Non-lytic release of enteric viruses in cloaked vesicles' was the topic of the keynote lecture by Nihal Altan-Bonnet, NIH, Bethesda, USA.

The Jean Cohen lecturer of this meeting was Polly Roy, London School of Hygiene and Tropical Medicine, who spoke on aspects of the replication cycle of bluetongue viruses, and how some of the data are similar to details of rotavirus replication.

This year, the triennial dsRNA virus symposium took place in Banff, Canada. It was very well organized by Maya Shmulevitz, University of Alberta, and Roy Duncan, Dalhousie University, and was attended by >150 participants. Besides the main lectures, there were ample opportunities for discussions at the two poster sessions (>70 posters, a selection of which was presented as 3 minute summaries to the full audience), during an afternoon of excursions to the impressive mountainous and canyon-rich landscape around Banff and during a Western barbecue, enriched by indigenous dancing presentations. Several introductory ‘educational sessions’ on the main topics of the meeting attracted much interest. The meeting was generously supported by industrial and academic sponsors.

The keynote address on ‘Advances in non-lytic release of non-enveloped viruses’ was given by Nihal Altan-Bonnet, National Institutes of Health. The talk concentrated on the recent discovery of en bloc transmission of enteric viruses (rotavirus, norovirus) in extracellular vesicles (EVs). The EVs remain intact during transmission. Compared to free virus particles, the transmission in vesicle-cloaked viruses appears to be 5 times more efficient (Santiana et al., 2018). Using suckling mice as a model and salivary gland-derived cell lines, it was discovered that enteric viruses can replicate in salivary glands and infect the mother's mammary gland through saliva in a retrograde way – in a backflow through suckling (Ghosh et al., 2022).

The main symposium consisted of six sessions devoted to specific topics.

1. The dsRNA virosphere: evolution and diversity

Ioly Kotta-Loizou, Imperial College London, reviewed the Polymycoviridae, an emerging virus family in the Riboviria realm containing 4 to 8 segments of dsRNA, which infect fungi, insects and humans (Kotta-Loizou et al., 2022). The diversity of mycovirus-mediated phenotypes depends on environmental factors of the virus-host interaction (Kotta-Loizou, 2021). Interestingly, naked dsRNA is infectious (Kanhayuwa et al., 2015); however, details of the replication have not been characterized.

Celeste Donato, Murdoch's Children Research Institute, reviewed the G/P diversity of species A rotaviruses (RVA) in Australia. Unusual rotavirus strains were detected through the Australian Rotavirus Surveillance Program between 2009 and 2019 and their sequences determined by Illumina MiSeq. In summary, the overall number of RVA isolates has decreased, but their diversity has increased, mainly due to the zoonotic transmission of animal G3, G6, G8 and G10 strains or their G/P encoding RNA segments (Donato et al., 2022).

Hayoto Harima, Tokyo University of Agriculture and Technology, described a bat-borne orthoreovirus isolated in Zambia which was found to be related to Nelson Bay Virus and considered as a potential cause of human respiratory infections (Harima et al., 2021). All post-juvenile bats were seropositive for this pteropine orthoreovirus.

Lisa Bono, Texas Technical University, assessed experimental data, mainly obtained with the cystovirus bacteriophage phi6, to explore how viruses may adapt to host change. During this process, termed niche expansion, some universal mutations emerged which were not consistently costly in terms of viral fitness (Bono et al., 2017; Bono et al., 2020).

Akthar Ali, University of Tulsa, described the newly classified Alternaviridae family that infect phytopathogenic fungi. These viruses carry 3 segments of dsRNA, the largest encoding the viral RdRp, and are candidate agents for use against pathogenic fungi that compromise agricultural crop production.

Aase Beathe Mikalsen, Norwegian University of Life Sciences, reviewed piscine myocarditis virus (PMCV), a Toti-like virus infecting shrimp, fish and insects. Such viruses were found to infect Atlantic salmons farmed on the West coast of Norway and causing myocarditis. The virus appears to undergo variations between and within individual hosts of the same disease outbreak (Sandlund et al., 2021).

José Castón, Centro Nacional de Biotecnología, described the molecular structure of the L-A dsRNA virus of Saccharomyces cerevisiae. The capsid consists of 60 homodimers in a T=1 icosahedral capsid with the dsRNA (of A structure) packaged in 3-4 concentric layers. Viral ssRNA and the viral RdRp are asymmetrically packaged, and the genomic dsRNA formed several filamentous layers with a single-spooled organization and loose genomic density (Luque et al., 2018; Mata et al., 2020).

2. Structure and function: virions, genomes and proteins

Jason Kaelber, Rutgers University, talked about quasispecies breaking within the order Reovirales, using Fakovirus, an insect virus in the Spinareoviridae family which possesses 9 genomic dsRNA segments, as an example. He suggested that members of the Reovirales have continuously lost RNA segments (starting from 12 segments in the Coltivirus genus) and that the number of RNA segments which could be packaged was constrained by their individual lengths. The polymerase complex of Fako virus was found in 10 different sites of the particle, despite there being only 9 RNA segments. This architecture may also be relevant for members of the Reoviridae with 10 and 11 RNA segments (Kaelber et al., 2020).

Minna Poranen, University of Helsinki, described the activation of semi-conservative intra-capsid transcription in the dsRNA virus phi6. She concentrated on the P7 protein which was found to be essential for transcription but not for dsRNA replication in the self-assembly system of phi6 (Poranen et al., 2001; Poranen et al., 2008; Ilca et al., 2019).

Kristen Ogden, Vanderbilt University Medical Center, expanded on the discovery that Fusion-Associated Small Transmembrane (FAST) proteins, which were initially described as products of the Orthoreovirus and Aquareovirus genera in the Spinareoviridae family (Duncan, 2019), have now also been found in species B rotavirus [RVB; Diller et al., 2019) and predicted to be expressed by RVG and RVI species. The NSP1 gene of RVB was found to contain two overlapping open reading frames, the smaller of which (NSP1-1) encodes a FAST protein. NSP1-1 gene expression from related RVG and RVI viruses in primate cells induced syncytia. The RVB NSP1-1 gene was engineered into the simian SA11 RVA by reverse genetics (RG), and this virus formed syncytia as well and replicated better in human colon cells than the parent virus. [A gain-of-function concern requires further exploration.]

David Gil-Cantero, Spanish National Research Council, described the organization of genomic dsRNA inside the capsid of Penicillium chrysogenum mycovirus. The viral genome consists of 4 segments of dsRNA encoding the RdRp, the capsid protein and two proteins of unknown function. Each RNA segment is encapsidated in separate similar particles with a relatively low genome density and an associated higher dsRNA mobility. The dsRNA is present in the A form, with 55% of the genome arranged directly below the capsid which is highly positively charged (Luque et al., 2018; Mata et al., 2020).

Using the provocative title ‘The battle between rotaviruses and humans at the atomic level’, Liya Hu, Baylor College of Medicine, reported on the structural interaction of rotavirus proteins with human cellular receptors. The full spectrum of glycan recognition of globally dominant RVAs was reviewed (Hu et al., 2018). Whereas species A and C rotavirus VP8* carry a galectin-like fold interacting with cellular glycans during cell entry, species B rotavirus VP8* lack such a structure. However, using the recently developed AlphaFold2 procedure for structure prediction and validation of the predicted structure by determining the 1.3 Å crystal structure, a novel protein fold was identified and shown to interact with N-acetyllactosamine residues of cellular glycans (Hu et al., 2022). This study demonstrated the combined power of AlphaFold2 structure prediction (Jumper et al., 2021) and crystallographic analysis and was considered to be applicable to other viruses. Structure analyses of the RVA capping enzyme VP3 showed it to form tetramers with each subunit responsible for the distinct steps of transcript capping (Kumar et al., 2020).

Gabriela Llauger, National Institute of Agriculture and Technology, took an interest in the viroplasm forming protein P9-1 of the Mal de Rio Cuarto virus, of the Fijivirus genus, which causes disease in maize and is transmitted by planthopper vectors. P9-1 dimerizes through hydrophobic interactions and forms decamers and dodecamers in insects. It is considered to be an analog of rotavirus NSP2; nanobodies against P9-1 are under development (Llauger et al., 2021).

Danica Sutherland, University of Pittsburgh, described the molecular details of the human neural orthoreovirus receptor Nogo-66 receptor 1 (NgR1). Unlike the JAM-A receptor which interacts with the sigma1 protein, NgR1 binds virus via its sigma 3 protein (Konopka-Anstadt et al., 2014). Interestingly, the murine homolog of NgR1 is dispensable for reovirus pathogenesis in the murine model, suggesting that additional receptors are likely to be involved (Aravamudhan et al., 2022).

Alexander Borodavka, University of Cambridge, reviewed the new concept of rotavirus viroplasms representing viral ribonucleoprotein (RNP) condensates and undergoing phase separation (PS) (Geiger et al., 2021). Similar phenomena have been found for the replication factories of influenza A virus, measles virus, respiratory syncytial virus, rabies virus and SARS-CoV-2. The structure of the viral NSP2 allows it to interact as a chaperone with viral ss(+)RNAs (Borodavka et al., 2017; Bravo et al., 2018), and its disordered charged C-terminal residues promote its displacement from the RNA (Bravo et al., 2021). In vitro reconstituted NSP2-NSP5 condensates absorb ssRNA, dsRNA and the viral RdRp. Interestingly, RNA structure probing using high-throughput SHAPE profiling revealed that NSP2 binding to ssRNA does not change the reactivity of the RNA significantly. However, mutational profiling analyses indicated a 5-fold rate increase in the presence of NSP2, with a larger effect on stems than on loops (Coria et al., 2022), suggesting that NSP2 binds across entire RNA, yet preferentially destabilizes smaller helical regions.

3. Virus replication: entry, exit and everything in between

John Parker, Cornell University, presented very comprehensive data on the host response to reovirus T1L-induced myocarditis in newborn mice, using spatiotemporal transcriptomics and single cell identification to integrate single cell data with the development of the infection in tissues (Mantri et al., 2021; McKellar et al., 2022). The detailed technology permitted to observe cell-specific viral tropism, innate immune responses at single cell level, and recruitment of various circulating immune cells. Endothelial cells were found to be infected first, followed by recruitment of cytotoxic T cells and a cascade ending in pyroptotic death. Inflammation of cardiomyocytes ended in tissue injury. Compared to wildtype virus, the reovirus T1L S4-K287T mutant was less virulent, replicating to lower levels in the heart and causing reduced inflammation

Justine Kniert, University of Alberta, spoke about spatiotemporal compartmentalization of reovirus assembly. Kinetic analysis of viral (+)ssRNA production and core synthesis vs outer capsid protein expression indicated that assembly of outer capsid proteins onto cores was temporally delayed and spatially segregated. All outer capsid proteins were associated with lipid droplets near the nucleus (Kniert et al., 2022).

Andrew Broadbent, University of Maryland, described the particle assembly of the birnavirus infectious bursal disease virus (IBDV). Viral factory (VF) formation was found to depend on VP3 and contained newly synthesized RNA. VFs were not membrane bound and could be dissolved by 1,6-hexanediol, consistent with them being subject to liquid-liquid phase separation (LLPS). VFs were distinct from paracrystalline arrays of virions in the cytoplasm (Reddy et al., 2022).

Marc Guimerà Busquets, The Pirbright Institute, analysed how infection with one orbivirus, the bluetongue virus strain 1 (BTV1), blocked superinfection with BTV8 during asynchronous infection. Blockage was time dependent in that it did not occur within 4 hours, but within 18 hours of asynchronous infection. At the blockage stage, no genome reassortment took place (Guimerà Busquets et al., 2021).

Alexander Borodavka, University of Cambridge, provided more details on phase separation (PS) of NSP5 and NSP2 during rotavirus replication. Upon mixing the viroplasm-forming proteins NSP2 and NSP5, biomolecular condensates formed at relevant low micromiolar concentrations. It turned out that liquid NSP5/NSP2 condensates are formed during the early infection stage but lose their fluidity late in infection (Geiger et al., 2021). Early infection stage condensates can be dissolved using 1,6-hexanediol or propylene glycol, releasing rotavirus transcripts and leading to a reduction of viral replication and a decrease of NSP5 hyperphosphorylation; this process was reversible. The transitions were characterized by microfluidics, single molecule FISH and DNA PAINT techniques. Viruses can be considered as models to interrogate details of phase transitions and phase separation in biology, and viral RNP condensates are becoming attractive targets for antiviral developments. These studies are benefitting from recent advances in machine learning (ML) approaches. Using ML, protein sequences of multiple viral strains can be classified into sub-variants of low- and high-propensity for phase-separation. These variants can be studied in detail, both in vitro, and using RG approaches.

Sarah Nichols, Wake Forest University, described a mutation at the C terminus of the rotavirus SA11 NSP2 protein (K294E), which decreased flexibility of this region and reduced replication. A recombinant virus rSA11-NSP2 K294E exhibited a 20-fold reduction in single cycle kinetics, producing smaller and more numerous viroplasms than observed with rSA11 wildtype. Co-expression of NSP2 K294E with NSP5 formed less viroplasm-like structures (VLS).

Catherine Eichwald, University of Zűrich, described the recruitment of the cytosolic TRiC chaperonin (TCP-1 Ring complex) to rotavirus viroplasms. TRiC had previously been shown to control orthoreovirus replication (Knowlton et al., 2018; Knowlton et al., 2021). A chemical TRiC inhibitor was found to prevent viral (-) strand RNA synthesis and the formation of TLPs, and led to the accumulation of empty viral particles.

Jacob Perry, Baylor College of Medicine, described the production of Ca2+ ‘puffs’ by rotavirus NSP4 expression in uninfected cells transfected with Ca2+ indicators. The ‘puffs’ occur at the onset of NSP4 expression and precede the production of intercellular Ca2+ waves (Chang-Graham et al., 2019; Chang-Graham et al., 2020). An NSP4 mutant (with a change in the viroporin domain, N77A) exhibited delays in puff production; other phenotypes of this mutant await exploration.

3.1. The Jean Cohen lecture

Polly Roy, London School of Hygiene and Tropical Medicine, was elected to be the Jean Cohen lecturer of the 2022 conference. Her talk, entitled ‘The replication cycle of bluetongue virus reveals its secrets, and those it shares with rotavirus’, summarized various aspects of her work on bluetongue virus (BTV) over the past 40 years, published in >350 scientific journal articles. While detailed description of her work would be of forbidding length in this context, the following results are considered to represent research highlights: the structure-function analysis of the viral RdRp, VP1 (Boyce et al., 2004; Wehrfritz et al., 2007); the establishment of a reverse genetics system for BTV (Boyce and Roy, 2007; Boyce et al., 2008); the in vitro reconstitution of BTV infectious cores (Lourenco and Roy, 2011); the elucidation of mechanisms of packaging of BTV RNA segments (Sung and Roy, 2014; Sung et al., 2019); and the development of a replication-deficient BTV vaccine (Celma et al., 2013; Celma et al., 2016). More recently, Dr Roy collaborated on the structure function analysis of rotavirus RdRp with the group of ZH Zhou (Ding et al., 2019).

4. Combatting and exploiting dsRNA viruses

John Patton, Indiana University, presented his expertise on the ‘Boundless flexibility of the rotavirus genome’. After reviewing several versions of rotavirus RG procedures (Kanai et al., 2017; Komoto et al., 2018; Philip et al., 2019; Philip and Patton, 2020) and the use of rotavirus RG as a vector system, he reported experiments attempting to explore the upper size limit of inserts (consisting of genes encoding fluorescent dyes, or the proteins of NoV, HEV, SARS-CoV-2, astrovirus, or others). Using the NSP3-based procedure (Philip and Patton, 2020), it was found that inserts of up to 1.1 kbp (adding to the size of the NSP3 gene) were tolerated in viable recombinants and were genetically stable upon serial passage in vitro. Recombinant rotaviruses with larger inserts had disadvantages: compared to the parent virus, they grew with lower growth kinetics and to lower titers, formed smaller plaques and were genetically unstable upon passage, loosing substantial parts of the large inserts. Vice versa, particular RNA segments could be significantly deleted as was also observed in natural RVA isolates (Taniguchi et al., 1996). The overall capacity of rotaviruses to accommodate heterologous inserts remains to be explored (Desselberger, 2020).

Takahiro Kawagishi, Stanford University, spoke about exploiting recombinant rotaviruses as intestinal vaccine vectors to express heterologous antigens from enteric pathogens. Using the NSP3-based expression vector (Philip and Patton, 2020) and immunodeficient mice (STAT-/-), the VP1 gene of a human norovirus (HuNoV) was expressed in vivo, and neutralization-equivalent antibodies (measured as reduction in genome equivalents) were observed. Concerns remain with regard to the genomic stability of the rRVs, and work is ongoing to reduce the size of the insert to that of the P domain of HuNoV VP1. Experiments have also been started to insert the gene encoding eltB, the enterotoxigenic E. coli heat labile toxin B subunit.

Francisca Cristi Munoz, University of Alberta, presented data on Super Virus 5 (SV5), a mammalian orthoreovirus, which has improved oncolytic activity in murine breast cancer models. The SV5 contains mutations in proteins sigma1, sigma3, lambda1, lambda2 and mu2 and possesses fewer sigma1 cell attachment molecules than wt virus. SV5 preferentially infects breast cancer cells, but exhibits lower capability of cell attachment, thus permitting further spread within tumor cells.

Asha Philip, Indiana University, described the development of a RG system for the Rotarix G1P[8] strain RIX4414 in order to exploit the potential to act as a vector for heterologous antigen expression and to advance the development of next-generation recombinant vaccines. The nt sequences of the RIX4414 strain were originally derived from GenBank, later determined from the virus. The system was established in MA104 cells using the SA11-based procedure. The NoV VP1 gene was inserted into the NSP3 gene as described (Philip and Patton, 2020). SARS-CoV-2 sequences (the receptor binding and S1 domains of the S gene) were also successfully inserted into the RIX4414 segment 7. Genetic stability will be tested by serial passage in vitro; animal experiments are planned, optimally in gnotobiotic piglets.

John Lewis, Entos Pharmaceuticals, reported on ‘Development of a COVID-19 DNA vaccine formulated with the fusion-associated small transmembrane protein proteolipid vehicle delivery system’. Based on knowledge of reovirus-encoded FAST proteins (Duncan, 2019), a proteolipid platform was developed that used microfluidics to mix a small amount of a chimeric FAST protein, co-expressed in a nanoplasmid expressing the S protein of SARS-CoV-2 or fragments thereof, with naturally occurring neutral lipids and a small amount of an ionizable lipid for packaging. Application of such constructs to mice (dose range 1-250 ug/mouse) or non-human primates induced strong neutralizing antibody responses, and a T cell-specific killing assay demonstrated S protein-specific T cell responses. Upon challenge in a hamster model, viral titres in the lung were decreased compared to non-vaccinated infected controls. A phase II clinical trial with this construct is currently ongoing.

Megan Stanifer, University of Florida, characterized interferons (IFNs) that prevent the spread of rotavirus in human intestinal epithelial cells. Wildtype rotavirus blocks the production of type I IFNs but elicits type III IFNs. Endogenous type III IFN was found to be decisive in limiting rotavirus infection of human intestinal cells (Doldan et al., 2022).

Jonathan Mandolo, Wellcome Trust Clinical Research Programme, described a prospective cohort study designed to examine the effects of breast milk on poor immune responses to rotavirus vaccination in Malawi. Results showed that maternal breast milk microbiota evolved over time, with an increase in microbiota diversity being negatively correlated with the immunogenicity of the rotavirus vaccine. This work expanded earlier data on host factors determining neonatal vaccine responses and rotavirus infections (Ramani et al., 2018; Parker et al., 2021).

Ming Tan, Cincinnati Children's Hospital Medical Center, reported on the development of a trivalent (VP8* subunits of P[4], P[6], and P[8]), pseudovirus S60 nanoparticle-based rotavirus candidate vaccine, which would be considered for parenteral use. According to preliminary data, high levels of neutralizing and cross-neutralizing antibodies were recorded in inoculated mice.

5. Rotavirus vaccines

Jacqueline Tate, CDC Centers for Disease Control and Prevention, summarized the impact of global application of rotavirus vaccines (Burnett et al., 2022; Hallowell et al., 2022; Glass et al., 2021). At present, universal rotavirus vaccination programs have been introduced in >100 countries worldwide. Global post-vaccine introduction surveillance has recorded a 40% reduction of pre-vaccine prevalence in morbidity and mortality, with the impact being higher in low-income than in high-income countries. Challenges remain: rotavirus infection is still a major cause of acute gastroenteritis (AGE); vaccine effectiveness (VE) is inversely related to mortality; there are numerous causes of decreased VE in low-income countries; withholding of breastfeeding does not improve the immune response; optimization of the number and strength of vaccine doses is required; possible improvement of VE by the addition of Zn and probiotics to vaccines has to be explored.

Venkata Raghava Mohan, Christian Medical College Vellore, assessed the impact and effectiveness of rotavirus vaccination in India. Two vaccine types are used (Rotavac (G9P[11]) and RotaSIIL (G1-G4, G8 P[8]). The VE was shown to be 45% with an overall impact of 20% (Nair et al., 2019).

Celeste Donato, Murdoch Children's Research Institute presented an update on the RV3-BB vaccine. Since its introduction (Bines et al., 2018), the vaccine has been found to be efficacious irrespective of histo-blood group antigen status (Boniface et al., 2020), and its formulation has been developed further (Kumar et al., 2021). Its performance is considered to be similar to that of the vaccines Rotarix, RotaTeq, Rotavac and RotaSIIL (Geard et al., 2022).

Vanessa Harris, Amsterdam University Medical Center, discussed the performance of oral rotavirus vaccines in low- and middle-income countries. The regulation of gut microbiota by viral infections through interferon signalling plays a major role (Wirusanti et al., 2022). 500 faeces from 122 infants in Ghana were collected and their viromes determined and analysed. Most frequently found viruses were picorna-, parvo-, reo-, anello- and cosaviruses. The presence of enteroviruses A and B seemed to prevent seroconversion following Rotarix vaccination (Kim et al., 2022). It was concluded that the gut virome can interfere with rotavirus vaccination take.

Sasirekha Ramani, Baylor College of Medicine, reported that human intestinal enteroids (HIE) can recapitulate interference between poliovirus and rotavirus vaccines. In analogy to population-based studies, co-administration of oral poliovirus (OPV) and oral rotavirus vaccines (ORV) to HIEs led to a reduced replication of rotavirus, most likely due to the fact that OPV replicates faster than ORV and due to exclusion of rotaviruses from OPV-infected cells. For further studies, genetically modified HIEs with disrupted innate immune response pathways will be used to identify factors mediating OPV interference.

Harry Greenberg, Stanford University School of Medicine, expanded on his excitement which the availability of RG for rotaviruses has raised: 1. It appears possible that rotaviruses can function as vector for immunization against NoV and numerous other enteric pathogens; 2. The genetic determinants of rhesus rotavirus (RRV) causing biliary obstructive jaundice/atresia in interferon alpha and gamma receptor 1 (IFNAR1, IFNGR1) -deficient mice can be explored; 3. The use of murine-like rRVs encoding a Nano-luciferase (NLuc) reporter can be applied to visual follow up of viral infection in animal models (Zhu et al., 2022).

James Platts-Mills, University of Virginia, analysed the burden of rotavirus disease in low- and middle-income countries in the post-vaccination era. Using defined parameters and reasonable assumptions, for the year 2034 a reduction in rotavirus-associated deaths of 49% is predicted (Kraay et al., 2022).

Chris Gast, PATH Center for Vaccine Innovation and Access, talked about a phase III clinical trial with the trivalent P2-VP8* vaccine which had been promising in phase I trials (Groome et al., 2020). A comparison with the Rotarix vaccine was carried out. Regrettably, the outcome led to a futility declaration, delaying further work on this vaccine candidate. Clinical samples acquired from the trial are still being explored to extract knowledge that might guide future trials.

Baoming Jiang, CDC Centers for Disease Control and Prevention, reviewed the development of an inactivated rotavirus vaccine (strain CDC-9 G1P[8] for intramuscular and intradermal inoculation (Moon et al., 2022). This strain has recently been analysed for binding broadly reactive antibody, using cryo-EM techniques (Jenni et al., 2022).

Lijuan Yuan, Virginia Polytechnic Institute and State University, reported on a mRNA-based trivalent P2-VP8* rotavirus candidate vaccine (Gebre et al., 2022) which was highly immunogenic and conferred protection in a gnotobiotic piglet model of human rotavirus infection and diarrhea (as described in Parreño et al., 2022).

6. Surviving and thriving: virus-host relationships

Anastasia Vlasova, Ohio State University, focussed on carbohydrate-mediated interactions between rotaviruses and intestinal epithelial cells. After reviewing established knowledge on rotavirus cellular receptors (Ramani et al., 2016; Amimo et al., 2021), she reported data on porcine intestinal enteroids (PIEs): different lineages of PIEs were found to support the propagation of different human RVAs to different degrees. 2-F-paracetyl fucose inhibited the replication of therotavirus Wa strain (G1P[8]); by contrast, treatment of PIEs with neuraminidase increased the growth of rotavirus Wa. Growth of RVC in PIEs depended on cellular cholesterol and sialic acid (Guo et al., 2022). Glycan-mediated interactions between bacteria, rotavirus and host cells were found to affect rotavirus growth and may lead to the development of beneficial probiotics (Michael et al., 2021; Raev et al., 2022). Amino acid substitutions of the hydrophobic region of VP4 (D385N, D393H) of the OSU strain are associated with attenuation (Guo et al., 2020). Work on an OSU rotavirus (G5P9[7])-based RG system is in progress.

Liliana Sánchez-Tacuba, Stanford University, spoke about the significance of VP4 of murine-like rotaviruses for host range restriction (HRR), using a suckling mouse model (Sánchez-Tacuba et al., 2022). Compared to the murine-like recombinant rotavirus, mono-reassortants carrying the VP4 gene of bovine and porcine rotaviruses but not of simian rotaviruses reduced diarrhea, and an analogous virus with human VP4 gene was also reduced in shedding. Analysis of rRVs harbouring chimeric VP8*-VP5* genes of bovine or murine origin led to the conclusion that VP8* was the determining factor of HRR. Altogether, the genetic origin of VP4 was identified to contribute, at least partially, to HRR.

Gudam Kwon, Seoul National University, reported on the analysis of transcription factors of the fungus Fusarium graminearum infected with F.g. virus 2. This virus is a mycovirus with 5 genomic segments of dsRNA. Some fungal host proteins were found to affect virus accumulation and transmission (Yu and Kim, 2020). More generally, cellular transcription factors were identified as being involved in various virus-host interactions (Li et al., 2019).

Francesca Scribano, Baylor College of Medicine, spoke about intercellular calcium dysregulation in cells infected with rotavirus and other viruses. Rotavirus infection and expression of NSP4 alone initiate the generation of intercellular Ca2+ waves with ADP functioning as a signalling molecule (Chang-Graham et al., 2019; Chang-Graham et al., 2020), thus proving that virus-host interactions involve uninfected cells. Such host cell signalling has also been recorded for infections with viruses of the Caliciviridae, Recoviridae and Picornaviridae families.

Sue Crawford, Baylor College of Medicine, explored rotavirus infections of HIEs by adapting them to growth in trans wells. Human rotaviruses were found to infect only basolaterally and be released apically. HIEs from all segments of the intestine could be infected. Rotavirus infection was found to be associated with fatty acid upregulation and lipid droplet biogenesis (Criglar et al., 2022). The ability of colonic HIEs to replicate rotaviruses was of particular interest.

Marie Hagbom, Linkoeping University, used large scale three-dimensional volumetric tissue imaging and brain stem histochemistry to show that rotavirus infection downregulates sympathetic nerve activity of the ileum of mice early in infection and that these changes are associated with increased intestinal movement and altered brain activities (Hellysaz et al., 2022).

Steeve Boulant, University of Florida, used a fluorescently labelled (UnaG) recombinant rotavirus (Philip et al., 2019) to follow rotavirus infection in vitro. Colonies of infected cells were observed early in infection. There was a correlation of the emergence of Ca2+ waves and rotavirus replication. Knocking out of the cellular ADP receptor decreased rotavirus replication (Stanifer and Boulant, 2020; commenting on Chang-Graham et al., 2020).

Megan Cole, The Pirbright Institute and University of Edinburgh, found that BTV interacts with the host's bacteria, lipopolysaccharides (LPS), and saliva. Bacteria (Bacillus subtilis) were found to enhance the infection of bovine blood-derived monocytes, possibly by stabilizing virions or by enhancing receptor binding. LPS were also found to enhance; this activity was lost after detoxification of LPS.

6.1. Poster presentations

The conference was enriched by >70 poster presentation which complemented the various topics addressed in oral sessions. A list of posters (First author, email address, affiliation, poster title) is attached [Supplementary Table S1]. Further details are available upon request from the conference organizers (Roy Duncan, Roy.Duncan@Dal.Ca, and Maya Shmulevitz, shmulevi@ualberta.ca).

6.2. Research progress within the last 4 years

Compared to the previous International dsRNA Virus Symposium (in Houffalize, Belgium, 2018), considerable progress can be recorded.

  • 1

    The availability of rapid next generation sequencing (NGS) techniques and further developments of bio-informatic algorithms has led to advancements in the classification and evolution of viruses.

  • 2

    The applicability of reverse genetics (RG) procedures to various dsRNA viruses has permitted the exploration of various basic questions of viral replication and pathogenesis and the exploration of dsRNA viruses (in particular rotaviruses) as vectors for heterologous expression of multiple enteropathogenic and systemically infecting microbes.

  • 3

    The combination of genetic engineering of dsRNA viruses by RG with the predictive power of AlphaFold2 is just at the beginning and will permit innovative exploration of mechanisms and functions of virus-host protein complexes.

  • 4

    The advent of intestinal enteroids of human and various other mammalian origins will initiate studies that complement pathogenicity data of animal models.

  • 5

    Progress in the recognition of mechanistic correlates of protection will enable the development of next generation vaccine candidates.

6.3. Next meeting

The 15th International Virus Symposium is planned to take place in Portugal in 2025 (details to be arranged).

Declaration of Competing Interest

I declare that I had no particular interests or prejudice when writing this report.

Acknowledgements

Comments and suggestions on a draft of this report by Alexander Borodavka, Sarah Caddy, José R Castón, Terence Dermody, Yuanji Ding, Roy Duncan, Mary K Estes, John Parker, and Maya Shmulevitz are gratefully acknowledged.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2022.199032.

Appendix. Supplementary materials

mmc1.docx (32KB, docx)

Data availability

  • The text reflects the proceedings of a conference.

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