Structural characterisation of the calicivirus entry pathway and its role in defining virulence

Caliciviruses are a family of viruses that cause diseases of global importance to human and veterinary medicine alike. Most notable of the human caliciviruses is the human norovirus, a virus that causes ~685 million cases of acute gastroenteritis each year worldwide; ~200 million cases are in children under 5 years, leading to 70,000-200,000 child deaths/year, mostly in developing countries. Every year human caliciviruses are estimated to cost ~$60 billion worldwide related to healthcare costs and lost productivity.

Caliciviruses of animals cause severe diseases, often with high morbidity and mortality, from usually fatal haemorrhagic disease in rabbits, enteritis in cattle to an acute respiratory disease in felids. While vaccines have been developed for the rabbit and feline caliciviruses, it is striking that the development of human caliciviral vaccines has lagged behind that of the animal caliciviruses. The host factors that affect calicivirus virulence remain to be identified and the pathogenesis of infection is poorly understood, although it is evident that viral evolution in both human and veterinary caliciviruses leads to the emergence of new variants.

Ultrastructural studies of human caliciviruses had been hampered for many years by the lack of suitable systems for the large-scale in vitro propagation of the viruses. Although recent breakthrough studies have revealed that several genotypes of human norovirus can be grown in human intestinal enteroids derived from stem cells, it is still not possible to prepare the high titres of the human viruses in vitro that are required for detailed structural studies. Elucidating the structural and biochemical properties of caliciviruses will play a key role in the structure-based design of therapeutics and will inform the development of novel caliciviral vaccines to impact health. Accordingly, we elected to analyse at atomic resolution the feline calicivirus (FCV) as a representative pathogenic calicivirus. We examined the early stage of calicivirus entry and infection, discovering that when the virus binds its receptor, a funnel-shaped structure forms on viral shell that likely allows the virus to initiate infection. In parallel with this discovery, we observed that emerging virulent FCV strains appear to bypass entry steps that are essential for infection with strains that cause respiratory disease; infection of cells with these and vaccine strains can be blocked by chloroquine, unlike virulent isolates.

This project brings together these two strands of research to examine the calicivirus-host interaction in avirulent and virulent strains of virus, comparing the two biological phenotypes and elucidating whether there are common structural determinants of caliciviral virulence. We will determine whether the novel structure observed in FCV is common to other caliciviruses by examining murine norovirus. The structure and function of the funnel-shaped structure observed on virions following receptor engagement will be modelled using lipid membranes to mimic viral uncoating and initiation of infection, enabling us to identify host-virus interactions that are critical for caliciviral entry.

We will compare the early entry requirements of a panel of pathogenic strains of calicivirus isolated from an outbreak of virulent systemic disease in felids, investigating the role that the viral entry process plays in determining viral pathogenicity. Chimaeric viruses will be generated to identify regions in the caliciviral capsid that confer the virulent phenotype.

This project will reveal fundamental insights into the early stages of infection with caliciviruses of humans and animals, facilitating structure-based approaches to the design of novel therapeutics and vaccines that will have a significant impact on human and animal health.

Caliciviruses cause diseases of global importance in human and veterinary medicine. Most notable of the human caliciviruses is norovirus, a ubiquitous virus that is difficult to control and causes a high global disease burden. Vaccines are essential for the effective control of norovirus, but technical challenges have hindered their development. Recently, we resolved the atomic structure of feline calicivirus (FCV), revealing the presence of a portal that forms following receptor engagement. We hypothesise that this structure mediates release of the genome from the endosome. Further, we have identified distinct biological phenotypes between avirulent and virulent strains of this virus using in vitro assays examining the early stages of infection. This project will investigate the early stages of infection with animal and human caliciviruses.

Firstly, we will compare the high-resolution structure of murine norovirus (MNV) with our solved structure of FCV, using cryoEM to assess conservation of structure and the conformational changes associated with receptor engagement across the Caliciviridae. We will determine the optimal conditions for viral entry and develop an assay to mimic endosomal escape, opening novel avenues for the screening of small molecule inhibitors of calicivirus entry, including human norovirus.

Secondly, we will compare the entry process for low and high virulence strains of virus, elucidating the link between viral entry and pathogenicity. An extended analysis of highly virulent caliciviruses will reveal the viral entry phenotypes of isolates for which whole genome sequences are available, permitting the mapping of potential virulence determinants. Candidate determinants will be confirmed using chimaeric viruses and mapped using site-directed mutagenesis and structure analysis.

Our findings will facilitate structure-based approaches to the design of novel therapeutics and vaccines for caliciviruses.

Viruses belonging to the Caliciviridae family are associated with severe diseases in humans and animals. The major human caliciviruses are the noroviruses (NoV) and the sapoviruses, which cause acute gastroenteritis and significantly impact human health. NoV infections cause 70,000-200,000 child deaths per year in developing countries. The very young, the elderly and immunocompromised individuals are at greatest risk from NoV infections; in the US, 19-21 million cases of human caliciviral gastroenteritis occur each year with ~800 deaths. Caliciviruses emerge in semi-closed settings; in 2018, 261 cases of NoV were confirmed at the Winter Olympics in Pyeongchang. Their stability in the environment leads to rapid spread as food contaminants; an outbreak of gastroenteritis in 2017 in Quebec was traced to contaminated raspberries. Important veterinary pathogens in this family include feline calicivirus (FCV) that causes "cat flu" and vesicular exanthema of swine virus, which infects pigs and marine mammals and is an important differential diagnosis for foot and mouth disease. Infections with European brown hare syndrome virus and rabbit haemorrhagic disease virus (RHDV) are often fatal. While there are no NoV vaccines, veterinary vaccines are available for the prevention of disease associated with infection with FCV and RHDV. These viruses share many characteristics with other members of the Caliciviridae; a comparative medicine approach is required to prevent diseases caused by other caliciviruses by vaccination. The obstacles to developing NoV vaccines include deficiencies in our knowledge of pathogenesis and immunity and the inability to produce high titre stocks. FCV represents a unique, readily accessible model that allows us to investigate fundamental features of caliciviral structure, biology, immunity and pathogenesis. Given the comparative value of these studies, our findings will inform the wider development of effective caliciviral vaccines and therapeutics. Ultimately, effective caliciviral vaccines ameliorating acute gastroenteritis in vulnerable people will be of significant benefit to human health.

We propose to exploit our recent cutting-edge findings, modelling the structural changes that occur during the early stages of calicivirus infection. We will extend our studies to include the murine norovirus as well as pathogenic calicivirus strains. Using our established proven model, we will compare a panel of well-characterised FCV isolates, examining in detail how structural changes in receptor binding relate to viral pathogenicity. Defining the structural changes following receptor binding will allow us to develop an in vitro assay that mimics the early stages of infection and will permit high throughput screening of potential antiviral therapies. To this end, we will explore routes to exploit our findings via the MRC technology transfer arm LifeArc and the GLAZgo Discovery Centre, a collaborative venture between the University and Astra Zeneca that aims to create better medicines.

The emergence of fatal, highly virulent systemic strains of FCV has been reported worldwide but the viral determinants of pathogenicity are unknown. Extending vaccine protection to these emerging strains will have a significant impact for veterinary vaccine manufacturers. We will translate our findings via our collaboration with Boehringer Ingelheim Animal Health, to improve existing FCV vaccines. Data collected by the UK pet food manufacturers' association show that the UK pet market is one of the largest in Europe; in 2018, 18% of households had cats. With 8 million owned cats in UK, of which 65% have had a primary kitten vaccination course (which includes vaccination against FCV), a strong case can be made for FCV vaccines contributing significantly to the UK economy. Ultimately, the value of these comparative studies for the human pharmaceutical market will be great.