1. Natural history

2. Aetiological agent

3. Clinical signs and lesions

4. Epidemiology

5. Virus life cycle

6. Mechanisms of resistance to RHD

7. Genetic diversity/RHDV evolution

7.1. Pathogenic RHDV

7.2. Non-pathogenic rabbit calicivirus

8. Host-virus co-evolution

9. Prevention, control and vaccination

10. Therapeutic applications of RHDV

11. Conclusions

12. List of abbreviations

13. Competing interests

14. Authors' contributions

15. Acknowledgements

16. References

In the 1980s, the European rabbit populations were devastated by a new viral disease characterised by being extremely lethal and highly contagious in both domestic and wild rabbits (Oryctolagus cuniculus). The first outbreak of this new disease, designated as rabbit haemorrhagic disease (RHD), was noticed in 1984 in the Jiangsu Province of the People's Republic of China within a group of commercially-bred Angora rabbits imported from Germany 1 . In less than a year, RHD killed 140 million domestic rabbits in China and spread over an area of 50 000 km² 1 2 . Korea was the next country to report RHD outbreaks which were associated with rabbit fur importation from China 3 . The disease then appeared in Europe and was first reported in Italy in 1986 4 from where it spread to the rest of Europe, becoming endemic in several countries. In the Iberian Peninsula, where European rabbits originated and where they constitute a key species of the ecosystem 5 , the first outbreaks date back to 1988 for Spain 6 and to 1989 for Portugal 7 and caused severe reduction of wild populations 8 9 . At the same time, domestic populations from several countries in North Africa experienced RHD outbreaks 10 . In the Americas, the first outbreaks were recorded in 1988 in Mexico following the importation of rabbit products from China 11 . Nevertheless, Mexico is currently the only country that has managed to successfully eradicate RHD with the last outbreak having occurred in 1992 11 . This successful eradication of the disease might correlate with the absence of natural populations of wild European rabbits. North America recorded the first outbreak only in 2000 and experienced a few additional outbreaks since then 12 . As the virus spread worldwide, naturally occurring RHD outbreaks were reported in geographically distant regions, such as Cuba, Uruguay and Reunion Island 13 14 .

RHD causes important economic losses in the rabbit meat and fur industry and has a significant negative ecological impact among wild rabbit populations and indirectly on its dependant predators 2 11 15 16 . In Australia and New Zealand, where the rabbit is considered an important agricultural pest, as well as a major threat to the endemic wildlife flora and fauna 17 18 , rabbit haemorrhagic disease virus (RHDV) was soon considered as an agent for rabbit control 19 . In 1991, a scientific research program was initiated in laboratory under quarantine measures to assess the host specificity and efficacy of the RHDV Czech reference strain (Czech V351) as a biocontrol agent. After approval of the Australian authorities, RHDV was released in the Wardang Island in Spencer Gulf, South Australia. Despite the rigorous quarantine measures, in 1995 RHDV escaped from the island, possibly transported by insects or air currents and reached the mainland 20 . In less than two years, it became established across southern Australia. The initial spread was estimated to be 50 km per week. In some areas a reduction of more than 95% of the wild rabbit populations was observed, particularly in the more arid regions 21 . In New Zealand, after a careful investigation on the benefits and risks of introducing RHDV, the government decided not to introduce the virus 19 . The virus was later illegally introduced by landholders 22 . Posterior characterisation of the New Zealand virus showed it to be similar to the Czech V351 strain introduced in Australia suggesting that it was imported from there 23 .

Nowadays, RHDV outbreaks still occur on almost all continents and cause significant mortality rates, being endemic in most parts of Europe, Asia, and parts of Africa, Australia and New Zealand. As a general trend, it seems that in areas where the European rabbit is historically present as wild populations, RHDV is also present and endemic. In contrast, in regions where the European rabbit is mainly present as a domestic or industrial animal, the occurrence of RHDV (epidemics or rare outbreaks) seems to be correlated with rabbit colony number and density.

Early efforts to classify RHDV were erratic, mostly due to its non-cultivable nature. Initially suspected to be a picornavirus 24 , a parvovirus 25 and a parvo-like virus 2 , it was finally assessed in the early 1990s as a member of the Caliciviridae family 26 27 28 29 30 .

The International Committee on Taxonomy of Viruses (ICTV) recognises four genera in the Caliciviridae family: Lagovirus, Vesivirus, Norovirus and Sapovirus. Three more genera were recently proposed as part of this family: Nabovirus or Becovirus 31 , Recovirus 32 and Valovirus 33 , but are not yet recognised by the ICTV. Caliciviruses infect a broad range of animals, including humans, and cause a variety of diseases, such as gastroenteritis by Norovirus and Sapovirus, haemorrhagic disease by Lagovirus, and vesicular lesions, respiratory infections and reproductive failure by Vesivirus. The Lagovirus genus comprises both RHDV and European brown hare syndrome virus (EBHSV), a virus first detected in Sweden in the early 1980s prior to the first RHDV outbreak 34 which affects hare species (Lepus europaeus and Lepus timidus). European brown hare syndrome (EBHS) is closely related to RHD with regards to clinical signs, pathological and histopathological alterations, mortality rates, virion morphology and antigenicity, but cross-species infection and cross-species protection could not be obtained in a reproducible way. Despite the similarities, RHDV and EBHSV represent distinct agents, infecting different species although causing similar diseases 35 36 37 38 39 40 .

As in other caliciviruses, RHDV virions are small sized (between 35-40 nm of diameter) and non-enveloped. The capsid, which forms the protein layer that protects the RNA molecule, is composed of 90 arch-like dimers of the capsid protein which form 32 cup-shaped depressions (calix in Latin for cup or chalice as the root for the family name Caliciviridae) arranged in a T = 3 icosahedral symmetry 41 42 . Each capsid monomer consists of a shell (S) domain which is buried and comprises the N-terminal connected by a hinge to the protruding (P) domain that encompasses the C-terminal region and is exposed on the surface 39 40 41 43 44 45 46 . The P domain can be further subdivided into the subdomains P1 (stem of arch) and P2 (top of arch) 47 . The subdomain P2, located at the most exposed region of the capsid, displays the greatest genetic variation. This variation is probably, at least in part, due to selection pressure because host antibodies recognise and target regions located in this subdomain 43 44 48 . In order to avoid this recognition and the inherent selective pressure, these regions tend to evolve faster 49 50 which results in an increase of the genetic variability and hence, of the antigenic variation. In addition, in noroviruses, the P2 domain has been shown to contain the carbohydrate-binding domain, contributing to several more conserved amino acids 51 52 53 . By analogy, this should also apply to RHDV.

Sporadically, in rabbits affected by subacute or chronic forms of RHD with long clinical courses, it is possible to detect a second type of virus particle, the RHDV core-like particles (CLP), also referred to as smooth particles or s-RHDV 35 . These particles are found in large amounts in the liver and spleen 54 and present unique characteristics when compared to RHDV particles: a smooth surface due to the lack of the cup-shaped depressions; a smaller diameter of 25-29 nm; a molecular weight of 28-30 kDa indicating that CLP correspond to the N-terminus (the buried shell domain) of the capsid; no haemagglutinating activity, most likely as the result of the absence of the C-terminus, but presenting reactivity with sera from RHDV convalescent rabbits and monoclonal antibodies directed towards the N-terminal part of the RHDV capsid 35 54 55 56 . CLP seem to be associated with the appearance of specific anti-RHDV IgM 54 . Indeed, these particles have been suggested to result from the degradation of the RHDV-IgM immune-complexes formed during the humoral response 54 . Although defective gene expression has been suggested to be at the genesis of CLP 55 , recent data indicate that CLP directly derive from intact virions with dissociated protrusion 45 .

RHDV virions contain the genomic RNA (gRNA) and an additional RNA species with 2.2 kb designated subgenomic RNA (sgRNA), which is collinear with the 3' end of the genomic RNA 26 57 . Subgenomic RNA usually contributes to the production of high levels of products required during the intermediate and late stages of infection (e.g. structural proteins) 58 . For RHDV, these comprise the capsid protein and VP10 59 60 61 . Both the genomic and subgenomic RNA are polyadenylated at the 3' end and at their 5' region they are covalently linked through a Tyr-21 residue to the VPg (virus genome-linked) protein 62 . The genomic RNA consists of a positive-sense single-stranded molecule of 7437 nucleotides consisting of two slightly overlapping open reading frames (ORF): ORF1, comprising nucleotides 10 to 7044 and ORF2, comprising nucleotides 7025 to 7378 26 . ORF1 encodes a large polyprotein of ca. 257 kDa 26 which is cleaved into the mature non-structural proteins and a major structural protein, the capsid protein, by post-translational proteolytic processing by a virus-encoded trypsin-like cysteine protease (Figure 1) 57 63 64 65 . Some of these proteins derive from larger precursors that result from further post-translational modifications of the precursor proteins 57 64 . The biological role of some of the non-structural proteins encoded by the genome of caliciviruses has been elucidated by relying on previous knowledge gathered from members of the closely related Picornaviridae family 26 64 65 . For RHDV, two proteins involved in the replication of the viral RNA, a helicase and an RNA-dependent RNA polymerase (RdRp), and a protease responsible for the proteolytic processing of the large polyprotein, have been characterised 63 66 67 . RdRp has been shown to also catalyse VPg uridylation 62 68 while a role in translation has been suggested for VPg [e.g. 69 ]. The function of the RHDV non-structural proteins p16, p23 and p29 remains to be assessed. VP10, a minor structural protein encoded by the 3'end of gRNA and sgRNA in a different reading frame (ORF2), was recently shown to increase the levels of virus replication and to promote apoptosis 70 . In addition, its ability to downregulate the expression of VP60 was demonstrated 71 . Together, this suggests that VP10 might regulate virus replication and virion release from infected host cells 71 .

The incubation period of the disease ranges between 1 to 3 days and rabbits usually succumb within 12 h to 36 h after the onset of fever (> 40°C). Depending on the clinical evolution of the disease, three different clinical courses can occur 38 72 . In the peracute form, infected animals show no clinical signs and die suddenly. Acute infections are accompanied by anorexia, apathy and congestion of the palpebral conjunctiva and neurologic symptoms such as opisthotonos, excitement, paralysis and ataxia may also be observed. There are occasionally some respiratory signs (tracheitis, dyspnea and cyanosis) and a foamy and bloody nasal discharge; lacrimation, ocular haemorrhages and epistaxis can also occur. Subacute forms of the disease present similar, but milder clinical symptoms and most rabbits survive. Rabbits experiencing subacute infections develop antibodies against RHDV which confer protection upon re-infection 73 . In addition, it has been reported that during an outbreak of RHD, a low percentage of rabbits may experience a chronic form of the disease with symptoms including a severe and generalised jaundice, anorexia and lethargy 35 . These animals tend to die 1-2 weeks later 54 , but animals that overcome the disease present a potent seroconversion 35 . Interestingly, this form of the disease has been shown to be associated with the presence of RHDV core-like particles 35 55 .

The liver, lung and spleen are the primary target tissues of RHDV. The major histopathological lesions found at necropsy are acute hepatitis due to liver cell loss as the result of RHDV-induced apoptosis, and splenomegaly 74 75 . Haemorrhages and congestions can be seen in several organs, particularly in the lungs, heart and kidneys, as a result of a massive disseminated intravascular coagulation (DIC) which is usually the cause of death 76 . Depletion of both B and T lymphocytes in the liver and the spleen accompanies the disease and accounts for an impairment of the immune response 72 77 and a fatal progression of the disease within 2-3 days. In contrast, resistant rabbits develop high titres of IgM (and then of IgA and of IgG) already at day 3 pi, thus presenting an effective humoral immune response 54 . Table 1 presents a summary of the histopathological alterations that can be observed upon RHDV infection.

The possible routes for transmission of the disease are the oral, nasal, conjunctival and parenteral, as blood-feeding insects have also been shown to be efficient mechanical vectors 72 78 . Transmission of RHDV may occur through direct contact with an infected animal, since infected rabbits may shed viral particles in their secretions and excretions 79 , or indirectly by means of fomites-contaminated food, bedding, water, clothing, cages and equipment 19 -or vector-borne transmission by scavenging mammals, birds and insects [e.g. 78 80 81 ]. The natural doors for viral entry have been suggested to be located in the upper respiratory and digestive tract 16 82 . In natural infections, the faecal-oral route is considered the preferential mode of transmission 10 11 .

In the field, carcasses of RHDV-infected rabbits may be a major source for viral spreading since the virus seems to be highly resistant and stable when exposed to harsh environmental conditions. Indeed, carcasses of RHDV-infected rabbits exposed to environmental conditions have been found to contain viable viral particles for up to three months 83 84 . This ability is paramount for the epidemiology of RHD and supports the importance of indirect routes in transmission. Environmental factors have also been suggested to impact on the effectiveness of RHD in rabbit populations [reviewed in 20 ]. Temperature and humidity seem to be the most important climate variables. Indeed, in Australia, mortality rates due to RHD are higher in arid and semi-arid inland areas than in moist coastal regions experiencing milder temperatures and the disease becomes active during the breeding season, peaks in early spring and is absent in the summer 85 . Climate variables might contribute to the geographic and seasonality observed for the RHDV outbreaks by affecting the abundance and activity of the vectors involved in RHDV transmission [reviewed in 20 ]. Other non-climatic factors have also been suggested to contribute to the variable pattern of the impact of RHD in rabbit populations such as the timing of the breeding season, the presence of a related and protective RHDV-like calicivirus in rabbit populations or the negative interaction of the myxomatosis outbreaks in the populations [reviewed in 86 ]. In addition, modelling studies indicated that population dynamics and spatial structure may greatly influence disease impact and host-virus co-evolution 87 88 .

Caliciviruses occur in a wide range of animals apart from rabbits, which include mustelids (minks and skunks), reptiles, cattle, felids (cats and cheetahs), dogs, humans, chimpanzees, pigs and sea mammals (sea lions, seals, walrus, whales, and dolphins), but they are usually restricted to their primary host and closely related species 89 . Indeed, rabbits and hares are the only hosts for the RHDV and EBHSV lagoviruses, respectively. Other leporid species have been shown not to be susceptible to RHDV 11 . Additionally, several non-host species from the Australian fauna, including domestic, feral animals and wildlife, were assessed for susceptibility to RHDV. No viral replication could be detected with an extensive panel of tests which included clinical observations, pathology, electron microscopy, virology and serology, reinforcing the idea that susceptibility to RHDV is restricted to the European rabbit (Oryctolagus cuniculus) 90 . Both subspecies of the European rabbit, O. c. cuniculus and O. c. algirus, seem equally susceptible to RHDV 91 . Interestingly, antibodies against RHDV had been found in animals that live in sympatry with rabbit populations infected with RHDV 81 92 93 and, more recently, RHDV RNA was isolated from sympatric wild micromammals opening the possibility of other species being involved in the epidemiology of the disease 94 .

In adult rabbits, the targets of the initial stages of the virus life cycle have been determined. Indeed, viral antigens are detected in the liver within the first hours following infection with RHDV with viral replication occurring in the cytoplasm of hepatocytes located mostly in centriacinar areas 30 74 95 96 97 98 . The number of infected hepatocytes clearly increases in the course of the disease, reaching a maximum between 36-48 h 95 96 97 . Detection of viral antigens in Kupfer cells has also been reported 74 98 associated with viral replication 98 . Extrahepatic presence of the virus has also been observed, but some discrepancies exist between the different studies since different techniques had been employed. Nevertheless, viral antigens have been detected in the spleen, in particular in the macrophages located in the red pulp 96 97 98 , kidney 96 , and alveolar macrophages in the lungs. It has been suggested that the presence of replicating virus in alveolar macrophages, which are in contact with the bloodstream, might be important for initial virus dissemination, and later when the virus reaches the liver, Kupfer cells may be important for spreading the infection into other organs 98 .

In contrast, viral dissemination in young resistant rabbits is far unclear. Viral antigens have been detected in hepatocytes from experimentally-infected 2-week old rabbits 99 , but most studies were only able to detect them in rabbits older than 4-weeks 96 100 . Viral antigens were found to be scattered and present in only a small percentage of cells. Nevertheless, this suggests that some hepatocytes in young (resistant) rabbits are able to support viral replication, but that major changes must occur in the liver to support a full infectious process. In addition, clearance of the virus seems to be extremely rapid as no viral antigens were detected after day 4 pi 100 . The presence of the virus in other organs has not been fully assessed.

However, as with most caliciviruses, understanding the interaction between RHDV and its host has been hampered by the lack of a suitable in vitro culture system. Consequently, studies on the pathogenesis of caliciviruses have relied on the ability of the capsid protein to self-assemble into virus-like particles (VLP) when expressed in insect cells. These particles have the advantage of being morphologically and antigenically indistinguishable from native virions, despite being devoid of viral RNA 59 101 102 103 104 105 . RHDV VLP strongly agglutinated human adult erythrocytes as the result of binding to glycolipid ligands present on the erythrocyte surfaces 72 . These ligands must develop with age since VLP did not agglutinate erythrocytes from human umbilical cords or foetuses presenting no agglutination 106 . Subsequent studies revealed that several caliciviruses use the carbohydrate moiety of host-cell histo-blood group antigens (HBGA) for attachment (e.g. ABH/O and Lewis antigens) initiating their replication cycle (Figure 2) 82 107 108 109 110 111 112 113 114 115 . Histo-blood group antigens are complex glycans either attached to proteins or lipids present on the surface of epithelial cells and erythrocytes, either as free oligosaccharides in biological fluids (milk, saliva, blood and intestinal contents). HBGA are formed by the sequential addition of monosaccharides to an oligosaccharide precursor chain attached to the cell glycans. This process, designated glycosylation, is catalysed by glycosyltransferase enzymes with specific substrate affinity and by a defined linkage 116 . Several genes encode the glycosyltransferases resulting in ABO, Lewis and secretor polymorphic phenotypes (Figure 3).

RHDV was shown to bind to the HBGA H type 2, A type 2 and B type 2 oligosaccharides 82 117 . These structures were shown to be present on the surface of the epithelial cells of the upper respiratory and digestive tracts that the virus first encounters when infecting the host and therefore where doors for virus entry are most likely located 16 82 . Synthesis of H type 2 requires the addition of a fucose residue, the minimal structural epitope 118 , in α1,2 linkage to a precursor. This reaction is catalysed by an α1,2-fucosyltransferase which in rabbits is encoded by three functional genes, Fut1, Fut2 and Sec1 that have undergone multiple events of gene conversion during evolution 119 . Synthesis of the A and B type 2 antigens involves the addition of either a N-acetylgalactosamine or a galactose residue in α1,3 linkage to the H type 2 trisaccharide in a reaction catalysed by an α1,3-N-acetylgalactosaminyltransferase or an α1,3-galactosyltransferase (A or B transferases), respectively. In rabbits, the ABO blood group locus is largely unresolved, but preliminary data suggest that at least 6 Abo genes exist in the genome, arranged in tandem (K Nyström and J Le Pendu, personal communication).

Following the attachment of RHDV to the cell surface, internalisation, by an unknown mechanism, and desencapsidation occur, leading to the release of the viral genome into the cytoplasm. The virus life cycle then proceeds to the translation of the polyprotein precursor encoded by the ORF1 of the viral genome through interaction with the host cellular machinery. The gRNA and the sgRNA covalently-linked VPg uses the cellular translation machinery, positioning the ribosome at the initiation codon AUG without ribosome scanning and initiating translation 69 120 . Post-translational proteolytic processing by the viral gRNA encoded protease cleaves the polyprotein precursor into the mature non-structural proteins and, in RHDV, into the capsid protein VP60 57 . The non-structural proteins, helicase and RdRp, then form a replication complex synthesising a complementary negative-sense RNA from the genomic RNA which is used as a template for the synthesis of gRNA and the sgRNA [reviewed in 121 ]. The resulting RNA can either be de novo translated or packaged into viral particles that will be released from the infected cell. The mechanism used by RHDV for dissemination of the viral progeny is still unclear, but the ability of VP10 to induce apoptosis may suggest a role in programmed cell death in virion release and dissemination 70 95 .

Translation of the RHDV ORF2 produces VP10 through a unique mechanism of reinitiation after termination of translation of the preceding major capsid protein 122 . This mechanism, although not fully understood, is dependent on the RNA sequence located upstream of the start/stop site, designated TURBS (termination upstream ribosomal binding site), but it is independent on the presence of the AUG initiation codon. Two critical motifs for VP10 expression have been identified within TURBS: motif 1, which is highly conserved among caliciviruses and shows complementarity with a short sequence in the 18S rRNA thus suggesting an interaction between the viral RNA and the ribosomal 18S rRNA, and motif 2, which is believed to be involved in the correct positioning of the ribosome at the translational start site 122 123 .

The lack of a cell culture system has been hampering the study of RHDV pathogenesis and, as a consequence, the mechanisms of resistance to the disease. The indirect strategies that have been employed for the study of the pathogenesis of RHDV allowed the identification of the HBGA H type 2 as an attachment factor for RHDV 82 . In humans, identification of alleles at the ABO, FUT2 and FUT3 loci that generate failure to express antigens recognised by different human Norovirus strains and that confer resistance to infection led to the search of such alleles in the α1,2-fucosyltransferase genes in adult rabbits 124 . This represented the first study on the genetic mechanisms underlying resistance to RHDV. A link between a rabbit allele at the α1,2-fucosyltransferase gene Sec1, that also intervenes in the H type 2 synthesis, and survival to a devastating RHDV outbreak was demonstrated 124 . This Sec1 allele encoded a weakly functional α1,2-fucosyltransferase, but was always found associated with Fut2 alleles coding for active enzymes that could compensate for the inability of Sec1 to synthesise H type 2. The authors hypothesised that this Sec1 allele was probably associated with a mutation located in the regulatory region of Fut2 which had compromised the Fut2 enzymes and, therefore, the synthesis of the virus' ligand. This result suggests that allelic variation in the α1,2-fucosyltransferase gene appears to have a significant role in resistance to RHDV. More recently, experimental challenge experiments indicated that at low virus titres, adult rabbits expressing low amounts of the HBGA ligands were less susceptible to the disease than animals expressing high amounts, although all animals were infected 117 .

One striking characteristic of the pathogenesis is that of resistance of young rabbits less than 2 months of age to RHD 72 . Indeed, kittens less than 3 weeks old are fully resistant, but when infected at an age of 4 weeks or older the mortality rates increase to reach, at about 9 weeks old, the rates observed for adult individuals 10 . Thus, the mechanisms of resistance to RHDV have also been studied in light of the differences observed between adult and young rabbits. Interestingly, in young-resistant rabbits the attachment factor H type 2 has been shown to be weakly expressed on the epithelial cells of the upper respiratory and digestive tracts, where primary infection by the virus is believed to occur 82 106 , which could explain their resistance to infection. Nevertheless, the reasons behind this differential expression have not yet been disclosed and the picture seems to be far more complicated. Indeed, and despite compelling evidence that supports a role of carbohydrates in facilitating infection by RHDV in epithelial cells of the upper respiratory and digestive tracts, other attachment factors or receptors must be playing a role at the epithelial level since low expression of the carbohydrate receptor at the doors of entry confers only partial protection against infection 117 . In addition, hepatocytes, the main cellular target for viral replication, have been shown not to express HBGA 82 and, in young rabbits, infection is accompanied by hepatic lesions due to virus replication as in adult individuals, although they tend to be more severe in 4 week old than in 2 week old rabbits 96 99 100 125 126 . This indicates the existence of at least additional hepatic cellular receptor(s) and that the genetic basis for the resistance mechanisms goes beyond the attachment of the virus to host cells through histo-blood group antigens. Immune response related-genes, either of the innate or the adaptive responses, are obvious candidate genes to be involved in the resistance mechanisms to RHDV and should deserve attention in future studies.

Differences in the innate immune response between RHDV-infected adult and young rabbits have also been observed 96 99 125 126 127 . Heterophils seem to be the predominant type of leukocyte in the liver inflammatory infiltrates in adult rabbits and are in close proximity with damaged hepatocytes probably being involved in the clearance of the dead cells. At variance, in young rabbits, this infiltrate is composed mostly by lymphocytes associated with undamaged and possibly antigen-presenting hepatocytes 127 and which are likely to mount a more effective and specific immune response than heterophils. In addition, in young rabbits only a small fraction of hepatocytes supports viral replication indicating that structural and functional changes have to occur in the liver to support RHDV replication 10 96 126 .

Development of enzyme-linked immunosorbent assays (ELISA) for the diagnosis of RHD 29 35 allowed an early determination of the importance of humoral immunity in the course of the disease. Indeed, animals experiencing subacute forms of RHDV that survived infection and that were later resistant upon re-exposure to RHDV where shown to present high levels of seroconversion 35 72 128 129 . A correlation between cELISA titres and protection has been established which is important for determination of the applicability of vaccination and to assess the current status of the disease 54 128 130 131 . Recovering rabbits present IgM titres that quickly reach a maximum within 2 weeks and then sharply decrease. IgA titres are more prolonged in time, but they also face a decrease. In contrast, IgG slowly increase and are able to persist for months. With regards to IgA, this suggests a mucosal response 128 . Passive immunization (serotherapy) was also shown to be effective in stopping RHD in a rabbit farm hit by an outbreak 132 , demonstrating the importance of humoral immunity in protection against RHD. In young rabbits, resistance has also been associated with the presence of maternal antibodies which are maintained during the period of life when they are considered RHD-resistant 128 . These are exclusively IgG acquired through the placenta in the last days of pregnancy and show a decline with age and body weight 19 . Additionally, if young rabbits are infected in their early life, they will become resistant when adult, suggesting that their immune system is capable of recognising the virus and producing an effective immune response that will confer long-term protection 133 . Therefore, humoral immunity clearly provides protection against RHDV when present 28 103 .

The virus-host dynamics result in a co-evolutionary process between the host resistance mechanisms and the virus escape mechanisms with attenuation of the virus and/or increase in resistance of the host. Therefore, the study of the host-virus co-evolutionary processes requires the analysis of each element of this dynamical pair simultaneously. Regarding the host, and in order to identify signatures of selection due to infectious agents, the natural history of this species should be considered. The fossil record suggests that the European rabbit originated in the Iberian Peninsula during the medium Pleistocene 189 190 191 192 and two morphologically differentiated subspecies have been distinguished: O. cuniculus algirus and O. cuniculus cuniculus 193 . O. c. algirus inhabits the southwestern Iberian Peninsula, while O. c. cuniculus is present in the northeastern Iberian Peninsula. These two subspecies diverged ~1.8 Mya [reviewed in 194 ] and then, by a post-glaciation expansion from the southwestern refugium to North and from the Northeastern to South or West, a contact zone was established. While the natural populations of O. c. algirus remained confined to the southwest of the Peninsula, the natural populations of O. c. cuniculus later expanded its range north towards France, likely after the last glacial peak 195 , where they still remain present. The expansion of these populations with successive bottleneck events caused a significantly lower genetic diversity of the wild French O. c. cuniculus populations compared to the Iberian populations 195 196 197 198 199 200 201 . The European rabbit gene pool has been manipulated by man through a recent single domestication event of French origin, and therefore, all domestic rabbits belong to the subspecies O. c. cuniculus [reviewed in 201 202 ]. Today, by man-mediated dispersal, the subspecies O. c. cuniculus can be found in Europe, Australia, New Zealand, North and South America, and North Africa. The gene pool of the European rabbit populations worldwide have been shaped differently by these events and this might have also interfered with the resistance mechanisms. Therefore, when studying the co-evolution between the European rabbits and RHDV, these events should be taken into account.

The introduction of myxoma virus in Australia was soon followed by an increase in genetic resistance in rabbits and the appearance of less virulent strains 203 . Recent field studies conducted in Australia suggest that RHDV also has become less effective in keeping wild rabbit numbers low and that in some populations rabbit numbers are returning to the pre-RHDV levels 204 . Laboratory challenges have confirmed this scenario when inoculation of rabbits from different Australian populations with the original introduced strain (Czech strain V351) failed to induce mortality or induced mortality rates lower than that which was expected 205 . In addition, in an Australian population, the frequency of host resistant phenotypes, i.e., phenotypes that confer a weak binding of the virus to the host HBGA that facilitate infection and thus provide protection to the host, has significantly increased 117 . A similar co-adaptation process seems to be occurring both in New Zealand 92 and in rabbit populations from Europe 117 . Indeed, Nyström et al. found that in a French wild rabbit population recovering from a major RHDV outbreak the frequency of resistant phenotypes increased among the survivors 117 . As with the Australian population, the resistant phenotypes are associated with weak viral binding. This indicates that the virus has contributed to select resistant hosts in accordance to the binding specificities of the circulating RHDV strains and gives further support for a role of the HBGA in the virus epidemiology and suggests that the virus is shaping the hosts' HBGA diversity.

The virus also seems to be evolving to overcome the host resistance mechanisms since significant mortalities are still observed in the field, at least in Australia. Evidence for this comes from the fact that in comparative trials in rabbits known to be resistant to infection with Czech 351, modern field strains appeared more virulent than the original released strain, suggesting they had evolved to keep pace with changes in rabbit resistance 205 . In addition, HBGA specificities of the strains that evolved in France from 1988 to 2009 progressively shifted, allowing preferential recognition of subgroups of animals that express distinct HBGA motifs, which suggests an adaptation to the host genetic diversity 117 .

In animals presenting subclinical or no clinical signs, passively acquired immunity has been shown to act successfully in emergency situations 132 . Indeed, this therapy, which is achieved by inoculation with a hyperimmune antiserum, confers short-term protection, preventing death. Nevertheless, passive immunization is ineffective on animals presenting clinical signs. Thus, as yet, no cure is available for RHDV-dying rabbits. Prevention and control of the disease through biosecurity and immunoprophylactic measures such as vaccination are, therefore, of utmost importance. Due to the lack of a cell culture system for efficient virus propagation, commercially available vaccines against RHDV are produced from tissue suspensions of experimentally infected rabbits, followed by chemical inactivation of the virus 132 177 206 . However, and to obviate the risks inherent to the manufacturing and use of this kind of vaccines (the use of infectious particles, the need for a safe disposal of contaminant residues, social concerns on animal welfare) the RHDV capsid protein has been tested in various studies as a subunit vaccine against RHD. Several heterologous expression systems or recombinant animal viruses have been developed to produce recombinant versions of the VP60 protein. The VP60 recombinant protein has been produced in Escherichia coli 140 ; insect cultured cells 59 101 102 103 104 ; yeast 207 208 ; plants 209 210 211 212 213 ; insect larvae 214 and recombinant animal-derived viruses 215 216 217 218 219 . Most of these systems were shown to be immunogenic and to confer protection against lethal doses of RHDV by eliciting a humoral response indicating that they are good substitutes for the tissue vaccines. Features such as low cost, high yields and ease of scaling up are amongst the most important factors for their commercial viability.

Although commercially available vaccines have proven effective in rabbitries, in wild rabbit populations vaccination campaigns are economically and logistically impracticable and their effects are considered insignificant 220 . Indeed, administration to wild rabbits implies capturing and handling of rabbits which by being a stress factor might increase the mortality rates 221 . In addition, this would need to be performed systematically since induced-immunity lasts no longer than 1 year 222 and efficacy has been shown to be dependent on several physiological parameters of the individuals 223 . Therefore, alternative approaches are being explored to overcome these limitations such as the development of vaccines with the capacity for horizontal transmission to ensure appropriate immunization of a relevant portion of the population 219 , vaccines that may be administered by the oral or nasal routes 105 208 213 217 219 224 225 or the construction of bivalent vaccines 216 226 . Nevertheless, as yet, none of these vaccines has been registered or is commercially available.

Biosecurity measures for control and prevention of RHD, including surveillance, sanitation, disinfection and quarantine, are of high importance to limit propagation and to ensure prevention of the disease in particular in the rabbit industry. In countries where RHDV circulates in wild rabbits and where eradication is not achievable, these measures might prevent large-scale infection in rabbitries. Thus, a careful and correct management of the RHDV outbreaks is always dependent on the epidemiological situation of the regions where they occur. In addition, a continuous monitoring of the viral evolution in the field is fundamental for the quick detection of new genetic and antigenic variants which might be determinant for the application of the most appropriate measures.

More recently, RHDV VLP have been considered as a mean for cancer and pathogen immunotherapies 227 228 229 230 231 232 . The capsid protein of RHDV spontaneously assembles into VLP which are morphologically and antigenically indistinguishable from native virions, but devoid of the viral RNA 101 . By genetic engineering, RHDV VLP have been shown to efficiently incorporate antigens that might be presented to immune cells and to elicit an adequate cell-mediated and humoral immune response 228 229 230 231 232 . In addition, RHDV VLP have the advantage of being easily produced and at low cost, and, by deriving from a non-human virus, they are not susceptible to pre-existing neutralising antibodies 228 , thus providing a reliable molecular tool for therapeutic applications.

RHDV has also been investigated for the study of virally-induced acute liver failure (ALF) in humans 233 234 235 236 237 238 239 as it fulfils several of the requirements to be a good animal model 233 . ALF is a condition characterised by severe liver injury, hepatic encephalopathy, coagulopathy and multiorgan failure, with viral infections (e.g. hepatitis A, B and E) and drug use (e.g. paracetamol overdose) amongst the commonest causes 240 . The hepatic lesions observed following infection by RHDV 99 resemble those caused by ALF in humans, but other physiologic, histological and biochemical alterations are also shared. Indeed, the hemodynamic changes, alterations in the intracranial pressure and histological alterations such as apoptosis observed in RHDV are common to ALF 74 233 241 242 . In addition, clinical symptoms such as prostration or convulsions observed on RHD are also observed in ALF 38 . RHDV has also been used for the study of therapeutic approaches for ALF 236 238 239 .

Despite the lack of an appropriate cell culture system, some light has been shed on several aspects of RHDV and RHD. Nevertheless, the host-parasite interactions established between RHDV and the European rabbit are still unclear. Indeed, the role of some of the proteins encoded by RHDV is still unknown and the emergence of RHDV as a pathogenic form has not yet been resolved. In order to clarify these gaps, an effort should be made in obtaining full genomic sequences, including for non-pathogenic strains as these might contribute to understand the pathogenesis of RHDV. As for the host, and in particular as for key factors of susceptibility and resistance, the rabbit genome project should be considered and used for the study of candidate genes. By using temporal samples, i.e., samples of rabbits collected before and after RHDV outbreaks, one might determine those candidate genes. Further studies on immunity to RHDV and on the related non-pathogenic viruses are also warranted for a better understanding of the host-pathogen relationships. In addition, the study of the closely related EBHSV and its host, might contribute to the understanding of the interplay between lagoviruses and leporid species. The possibility that RHDV might be used as a model for the study of other calicivirus infections, in particular in view of its non-pathogenicity for humans, as well as for the development of anti-cancer and pathogen therapies transforms it into a valuable research molecular tool.

RHDV: rabbit haemorrhagic disease virus; RHD: rabbit haemorrhagic disease; EBHSV: European brown hare syndrome virus; EBHS: European brown hare syndrome; gRNA: genomic RNA; sgRNA: subgenomic RNA; VPg: virus genome-linked protein; ORF: open reading frame; RdRp: RNA-dependent RNA polymerase; DIC: disseminated intravascular coagulation; pi: post-infection; VLP: virus-like particles; HBGA: histo-blood group antigens; TURBS: termination upstream ribosomal binding site; RCV: rabbit calicivirus; TMRCA: Time to Most Recent Common Ancestor; ALF: acute liver failure.

The authors declare that they have no competing interests.

JA performed a study on the available literature on the subject, analysed the retrieved information and wrote the manuscript. WvdL, JLP and PJE revised the manuscript critically according to their own area of expertise. All authors read and approved the final manuscript.