First Detection of Benign Rabbit Caliciviruses in Chile.

Pathogenic lagoviruses (Rabbit hemorrhagic disease virus, RHDV) are widely spread across the world and are used in Australia and New Zealand to control populations of feral European rabbits. The spread of the non-pathogenic lagoviruses, e.g., rabbit calicivirus (RCV), is less well studied as the infection results in no clinical signs. Nonetheless, RCV has important implications for the spread of RHDV and rabbit biocontrol as it can provide varying levels of cross-protection against fatal infection with pathogenic lagoviruses. In Chile, where European rabbits are also an introduced species, myxoma virus was used for localised biocontrol of rabbits in the 1950s. To date, there have been no studies investigating the presence of lagoviruses in the Chilean feral rabbit population. In this study, liver and duodenum rabbit samples from central Chile were tested for the presence of lagoviruses and positive samples were subject to whole RNA sequencing and subsequent data analysis. Phylogenetic analysis revealed a novel RCV variant in duodenal samples that likely originated from European RCVs. Sequencing analysis also detected the presence of a rabbit astrovirus in one of the lagovirus-positive samples.

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Interventions for the long-term prevention of hereditary angioedema attacks.

BACKGROUND: Hereditary angioedema (HAE) is a serious and potentially life-threatening condition that causes acute attacks of swelling, pain and reduced quality of life. People with Type I HAE (approximately 80% of all HAE cases) have insufficient amounts of C1 esterase inhibitor (C1-INH) protein; people with Type II HAE (approximately 20% of all cases) may have normal C1-INH concentrations, but, due to genetic mutations, these do not function properly. A few people, predominantly females, experience HAE despite having normal C1-INH levels and C1-INH function (rare Type III HAE). Several new drugs have been developed to treat acute attacks and prevent recurrence of attacks. There is currently no systematic review and meta-analysis that included all preventive medications for HAE. OBJECTIVES: To assess the benefits and harms of interventions for the long-term prevention of HAE attacks in people with Type I, Type II or Type III HAE. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 3 August 2021. SELECTION CRITERIA: We included randomised controlled trials in children or adults with HAE that used medications to prevent HAE attacks. The comparators could be placebo or active comparator, or both; approved and experimental drug trials were eligible for inclusion. There were no restrictions on dose, frequency or intensity of treatment. The minimum length of four weeks of treatment was required for inclusion; this criterion excluded the acute treatment of HAE attacks. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Our primary outcomes were 1. HAE attacks (number of attacks per person, per population) and change in number of HAE attacks; 2. mortality and 3. serious adverse events (e.g. hepatic dysfunction, hepatic toxicity and deleterious changes in blood tests). Our secondary outcomes were 4. quality of life; 5. severity of breakthrough attacks; 6. disability and 7. adverse events (e.g. weight gain, mild psychological changes and body hair). We used GRADE to assess certainty of evidence for each outcome. MAIN RESULTS: We identified 15 studies (912 participants) that met the inclusion criteria. The studies included people with Type I and II HAE. The studies investigated avoralstat, berotralstat, subcutaneous C1-INH, plasma-derived C1-INH, nanofiltered C1-INH, recombinant human C1-INH, danazol, and lanadelumab for the prevention of HAE attacks. We did not find any studies on the use of tranexamic acid for prevention of HAE attacks. All drugs except avoralstat reduced the number of HAE attacks compared with placebo. For breakthrough attacks that occurred despite prophylactic treatment, intravenous and subcutaneous forms of C1-INH and lanadelumab reduced attack severity. It is not known whether other drugs have a similar effect, as the severity of breakthrough attacks in people taking drugs other than C1-INH and lanadelumab was not reported. For quality of life, avoralstat, berotralstat, C1-INH (all forms) and lanadelumab increased quality of life compared with placebo; there were no data for danazol. Four studies reported on changes in disability during treatment with C1-INH, berotralstat and lanadelumab; all three drugs decreased disability compared with placebo. Adverse events, including serious adverse events, did not occur at a rate higher than placebo. However, serious adverse event data and other adverse event data were not available for danazol, which prevented us from drawing conclusions about the absolute or relative safety of this drug. No deaths were reported in the included studies. The analysis was limited by the small number of studies, the small number of participants in each study and the lack of data on older drugs, therefore the certainty of the evidence is low. Given the rarity of HAE, it is not surprising that drugs were rarely directly compared, which does not allow conclusions on the comparative efficacy of the various drugs for people with HAE. Finally, we did not identify any studies that included people with Type III HAE. Therefore, we cannot draw any conclusions about the efficacy or safety of any drug in people with this form of HAE. AUTHORS' CONCLUSIONS: The available data suggest that berotralstat, C1-INH (subcutaneous, plasma-derived, nanofiltered and recombinant), danazol and lanadelumab are effective in lowering the risk or incidence (or both) of HAE attacks. In addition, C1-INH and lanadelumab decrease the severity of breakthrough attacks (data for other drugs were not available). Avoralstat, berotralstat, C1-INH (all forms) and lanadelumab increase quality of life and do not increase the risk of adverse events, including serious adverse events. It is possible that danazol, subcutaneous C1-INH and recombinant human C1-INH are more effective than berotralstat and lanadelumab in reducing the risk of breakthrough attacks, but the small number of studies and the small size of the studies means that the certainty of the evidence is low. This and the lack of head-to-head trials prevented us from drawing firm conclusions on the relative efficacy of the drugs.

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they 'chaperone' p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

Frequent intergenotypic recombination between the non-structural and structural genes is a major driver of epidemiological fitness in caliciviruses.

The diversity of lagoviruses (Caliciviridae) in Australia has increased considerably in recent years. By the end of 2017, five variants from three viral genotypes were present in populations of Australian rabbits, while prior to 2014 only two variants were known. To understand the evolutionary interactions among these lagovirus variants, we monitored their geographical distribution and relative incidence over time in a continental-scale competition study. Within 3 years of the incursion of rabbit haemorrhagic disease virus 2 (RHDV2, denoted genotype GI.1bP-GI.2 [polymerase genotype]P-[capsid genotype]) into Australia, two novel recombinant lagovirus variants emerged: RHDV2-4e (genotype GI.4eP-GI.2) in New South Wales and RHDV2-4c (genotype GI.4cP-GI.2) in Victoria. Although both novel recombinants contain non-structural genes related to those from benign, rabbit-specific, enterotropic viruses, these variants were recovered from the livers of both rabbits and hares that had died acutely. This suggests that the determinants of host and tissue tropism for lagoviruses are associated with the structural genes, and that tropism is intricately connected with pathogenicity. Phylogenetic analyses demonstrated that the RHDV2-4c recombinant emerged independently on multiple occasions, with five distinct lineages observed. Both the new RHDV2-4e and -4c recombinant variants replaced the previous dominant parental RHDV2 (genotype GI.1bP-GI.2) in their respective geographical areas, despite sharing an identical or near-identical (i.e. single amino acid change) VP60 major capsid protein with the parental virus. This suggests that the observed replacement by these recombinants was not driven by antigenic variation in VP60, implicating the non-structural genes as key drivers of epidemiological fitness. Molecular clock estimates place the RHDV2-4e recombination event in early to mid-2015, while the five RHDV2-4c recombination events occurred from late 2015 through to early 2017. The emergence of at least six viable recombinant variants within a 2-year period highlights the high frequency of these events, detectable only through intensive surveillance, and demonstrates the importance of recombination in lagovirus evolution.

Calicivirus Non-structural Proteins: Potential Functions in Replication and Host Cell Manipulation.

The Caliciviridae are a family of viruses with a single-stranded, non-segmented RNA genome of positive polarity. The ongoing discovery of caliciviruses has increased the number of genera in this family to 11 (Norovirus, Nebovirus, Sapovirus, Lagovirus, Vesivirus, Nacovirus, Bavovirus, Recovirus, Salovirus, Minovirus, and Valovirus). Caliciviruses infect a wide range of hosts that include fishes, amphibians, reptiles, birds, and marine and land mammals. All caliciviruses have a genome that encodes a major and a minor capsid protein, a genome-linked viral protein, and several non-structural proteins. Of these non-structural proteins, only the helicase, protease, and RNA-dependent RNA polymerase share clear sequence and structural similarities with proteins from other virus families. In addition, all caliciviruses express two or three non-structural proteins for which functions have not been clearly defined. The sequence diversity of these non-structural proteins and a multitude of processing strategies suggest that at least some have evolved independently, possibly to counteract innate and adaptive immune responses in a host-specific manner. Studying these proteins is often difficult as many caliciviruses cannot be grown in cell culture. Nevertheless, the study of recombinant proteins has revealed many of their properties, such as intracellular localization, capacity to oligomerize, and ability to interact with viral and/or cellular proteins; the release of non-structural proteins from transfected cells has also been investigated. Here, we will summarize these findings and discuss recent in silico studies that identified previously overlooked putative functional domains and structural features, including transmembrane domains that suggest the presence of viroporins.

Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers.

Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-ß inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that-like many other caliciviruses-does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.

Harnessing therapeutic viruses as a delivery vehicle for RNA-based therapy.

Messenger RNA (mRNA) and microRNA (miRNA)-based therapeutics have become attractive alternatives to DNA-based therapeutics due to recent advances in manufacture, scalability and cost. Also, RNA-based therapeutics are considered safe since there are no risk of inducing genomic changes as well as the potential adverse effects would be only temporary due to the transient nature of RNA-based therapeutics. However, efficient in vivo delivery of RNA-based therapeutics remains a challenge. We have developed a delivery platform for RNA-based therapeutics by exploiting the physicochemical properties of enveloped viruses. By physically attaching cationic liposome/RNA complexes onto the viral envelope of vaccinia virus, we were able to deliver mRNA, self-replicating RNA as well as miRNA inside target cells. Also, we showed that this platform, called viRNA platform, can efficiently deliver functional miRNA mimics into B16.OVA tumour in vivo.

Calicivirus RNA-Dependent RNA Polymerases: Evolution, Structure, Protein Dynamics, and Function.

The Caliciviridae are viruses with a positive-sense, single-stranded RNA genome that is packaged into an icosahedral, environmentally stable protein capsid. The family contains five genera (Norovirus, Nebovirus, Sapovirus, Lagovirus, and Vesivirus) that infect vertebrates including amphibians, reptiles, birds, and mammals. The RNA-dependent RNA polymerase (RdRp) replicates the genome of RNA viruses and can speed up evolution due to its error-prone nature. Studying calicivirus RdRps in the context of genuine virus replication is often hampered by a lack of suitable model systems. Enteric caliciviruses and RHDV in particular are notoriously difficult to propagate in cell culture; therefore, molecular studies of replication mechanisms are challenging. Nevertheless, research on recombinant proteins has revealed several unexpected characteristics of calicivirus RdRps. For example, the RdRps of RHDV and related lagoviruses possess the ability to expose a hydrophobic motif, to rearrange Golgi membranes, and to copy RNA at unusually high temperatures. This review is focused on the structural dynamics, biochemical properties, kinetics, and putative interaction partners of these RdRps. In addition, we discuss the possible existence of a conserved but as yet undescribed structural element that is shared amongst the RdRps of all caliciviruses.

Characterization of Mutational Tolerance of a Viral RNA-Protein Interaction.

Replication of RNA viruses is generally markedly error-prone. Nevertheless, these viruses usually retain their identity under more or less constant conditions due to different mechanisms of mutation tolerance. However, there exists only limited information on quantitative aspects of the mutational tolerance of distinct viral functions. To address this problem, we used here as a model the interaction between a replicative cis-acting RNA element (oriL) of poliovirus and its ligand (viral protein 3CD). The mutational tolerance of a conserved tripeptide of 3CD, directly involved in this interaction, was investigated. Randomization of the relevant codons and reverse genetics were used to define the space of viability-compatible sequences. Surprisingly, at least 11 different amino acid substitutions in this tripeptide were not lethal. Several altered viruses exhibited wild-type-like phenotypes, whereas debilitated (but viable) genomes could increase their fitness by the acquisition of reversions or compensatory mutations. Together with our study on the tolerance of oriL (Prostova et al., 2015), the results demonstrate that at least 42 out of 51 possible nucleotide replacements within the two relevant genomic regions are viability-compatible. These results provide new insights into structural aspects of an important viral function as well as into the general problems of viral mutational robustness and evolution.