Leporids’ Emerging Diseases as a Threat to Biodiversity

Abstract

Wild leporids have been gaining interest and prominence in the scientific and social community worldwide. While endangered of extinction in its native territory, the Iberian Peninsula, where it has a key role in the Mediterranean ecosystems, the European rabbit (Oryctolagus cuniculus) is considered a plague in Australia, due to the great economic and ecological consequences of its presence in the territories. The impact of viral diseases on the Leporidae family’ members, namely on the European rabbit, has been largely recognized worldwide since the early 50s, due to the emergence of myxomatosis and, from the mid-80s onwards, due to the emergence of rabbit haemorrhagic disease virus 1 and 2. More recently, in 2018, a recombinant myxoma virus emerged with the ability to infect and cause severe disease in the Iberian hare (Lepus ganatensis). Also, a new gammaherpesvirus was described in Iberian hares, associated with myxoma virus infections. In this chapter, we revise the main viral infectious treats to the native leporids of the Iberian Peninsula. The recovery of the European rabbit populations, as well as of other leporid species around the world, is currently a major challenge for the scientific and social communities and policy-makers. If we fail, the ripple effects on the trophic web will be so dramatic that are likely to be unrecoverable.

1. Introduction

With the earliest known fossil ancestor identified in Mongolia 55 million years ago [1], Leporidae is the biggest family of order Lagomorpha and includes rabbits and hares (and also the jackrabbits), divided into 11 genera comprising 62 species, characterized, among other features, for the 28 teeth (incisor 2/1, canine 0/0, premolar 3/2, molar 3/3) (Figure 1) [2].

Figure 1.

Genera in families Ochotonidae (pikas) and Leporidae (leporids). The scientific names of species with conservation status of vulnerable, threatened or critically endangered by the IUCN are presented.

Together with family Ochotonidae that includes the pikas, Leporidae family constitute the mammalian Lagomorpha order. The 11 genera within the Leporidae family comprise genus Caprolagus (1 species), genus Lepus (32 species), genus Pronolagus(3 species), genus Sylvilagus (17 species), genus Brachylagus (1 species), genus Bunolagus (1 species), genus Nesolagus (1 species), genus Oryctolagus (1 species), genus Pentalagus (1 species), genus Poelagus (1 species) and genus Romerolagus (1 species).

Except for Antarctica and Australia, all continents have indigenous species of Leporidae. Genus Lepus is the most speciose genus and has a worldwide distribution [3], despite only six of these species inhabit Europe.

The European rabbit was introduced to most of Oceania as to other islands. The European rabbit, Oryctolagus cuniculus (Linnaeus, 1958), is native to the Iberian Peninsula and the south of France, while genus Sylvilagus is widely distributed across North, Central and South America, although most species are confined to specific regions.

Twelve species, namely 11 from Lepus genus and the European rabbit, have seen their geographic distribution expanded by human translocations since the year 1400 BC [4].

Leporids are key species in many ecosystems [5, 6] due to their role as primary consumers transforming the vegetal protein in high-quality animal protein [7, 8, 9], impacting directly and indirectly in the ecosystems. Its variable weight range, from Oryctolagus cuniculus algirus with less than 1 kg to Lepus europaeus with ~5 kg of weight, offers the ideal biomass intake for many predators from small, such as the genet, to big sizes, such as the lynx and eagles.

The reduction of native species can cause important changes in the structure and function of natural ecosystems affecting directly its intermediate and top predators, and indirectly other species belonging to the same and other linked web chains.

Regarding their behaviour, rabbits and hares are crepuscular, being most active at sunrise and sunset, during the twilight hours, being ideal prey species for predators with the same habits.

In native countries, these species have a huge impact on plant community composition and dissemination, slowing down some invasive grass species. However, in some cases, wild leporids have a great impact in the recruitment of young trees, forest restoration and maintenance of important native vegetation, while still having a positive impact on the survival of plants in hostile environments, being important in both mechanisms of endozoochory or epizoochory [10, 11, 12, 13].

European rabbit (O. cuniculus) is the most paradigmatic and well-studied leporid being a endemic species of Iberian Peninsula, consumed by more than 40 terrestrial and aerial carnivore species providing the primary biomass source for many of them [14, 15, 16], namely the paradigmatic Iberian lynx and Spanish Imperial eagle that include wild rabbits in about 90% of the diet [17]. Leporids are even preyed upon by very unusual predators, from light-weight terrestrial birds, such as the Greater Roadrunner (Geococcyx californianus) or the American red squirrel (Tamiasciurus hudsonicus) [18, 19].

High abundances of O. cuniculus may also have cascading effects by promoting the presence of top predators, such as the Iberian lynx, which regulate mesopredators, such as the Egyptian mongoose, Herpestes ichneumon, either via intraguild predation or by consuming their main prey [20].

The impacts of European rabbit in the Iberian Peninsula (Portugal and Spain) are the most notorious and well-studied, taking into account the essential effects of wild rabbit on these territories. Belonging to one of the 34 global ecological hotspots—the western corner of the Mediterranean Basin hotspot—second only to the tropics in importance [21], Iberia contains the same plant richness (30,000 taxa) of all tropical Africa (four times larger) and 10.8 species/1000 km², higher than China, Zaira, India and Brazil [22]. The reduction of leporids in this type of rich territories has brutal knock-on effects with tropic cascades, leading to threatens to all the trophic chains [16, 23]. The deregulation of the trophic chain also leads to the deregulation of the biomass sources of predators with effects on the proximity of predators to humans and with an increase in conflicting events between fauna and man. The principal events of viral threats are shown in the Figure 2.

Figure 2.

Major virus emergencies impacting wild leporids. LeHV, leporid herpesvirus; MYXV, myxoma virus; ha-MYXV, natural recombinant myxoma virus; GI.1, Lagovirus europaeus genogroup I, genotype 1 (RHDV); GI.2, Lagovirus europaeus genogroup I, genotype 2 (RHDV2/RHDVb).

Many factors account for the abrupt reduction of wild leporids in the last decades including habitat loss to agriculture or intensive forest regimes, habitat disturbance and fragmentation, intensification of agriculture with monoculture farming, excessive hunting pressure, excessive predation, climate exchanges (thermal limits, vegetation, rainfall) and soil type (impacting on burrowing and growing of major food species) [16, 24, 25]. In fact, all these parameters insidiously and chronically influence the wild rabbit populations, which are then subject to epidemic outbreaks, more visible and better studied, of various diseases, particularly those of viral aetiology.

Paradoxically, European rabbit (O. cuniculus) is also included on the ‘100 of the world’s worst invasive alien species’ list, and much scientific attention has been paid to this species as it has led to significant economic and ecological losses [26, 27].

Their high reproduction rates may be their main strategy to cope with predation, which in some cases can cause juvenile mortality of up to 90% [28, 29]. Most leporids have multiple litters per year, with litter sizes varying from 1 to 11 individuals, and each female producing between 10 and 45 young per year [6, 30, 31].

The presence of leporid species can also constitute a human-wildlife health hazard, as they act as natural reservoirs of many zoonotic diseases including, among many others, tularaemia, Lyme borreliosis and Crimean-Congo haemorrhagic fever [32].

In Argentinean Patagonia, the prevalence of Fasciola hepatica in L. europaeus is sufficient to maintain a viable wild reservoir of this disease [33, 34]. Sylvilagus floridanus is an effective vector of the West Nile virus, a threat to several vertebrate species [35], mainly human, horses and birds, and carries dermatophyte fungi that affect humans [36]. In its exotic geographic range in Italy, S. floridanus hosts many transmittable parasites and is an asymptomatic carrier of myxomatosis and pseudo-tuberculosis, directly affecting native leporids [37].

As obligatory intracellular parasites, viruses have evolved and adapted to their hosts in order to survive and produce a viable progeny. To do so, viruses need to take over the cellular metabolism from which they are totally dependent. Highly complex viruses, such as myxoma virus, can evade the host defence mechanisms by modulating the immune response through viral proteins.

In most occasions, viruses cause asymptomatic subclinical or mild infections in their natural hosts (reservoirs), a way to guarantee a continuous source and spread of virus for new replication cycles. Viral infections tend to be less aggressive in their natural host species, to which viruses are well adapted, than in introduced species, with no history of contact with (and adaptation to) the virus [38]. A good example of this is provided by myxoma virus, which produces a small benign fibroma in its natural host the Sylvilagus spp., the American rabbit (myxoma virus natural host or reservoir), but a highly pathogenic disease in the European rabbit (Oryctolagus cuniculus).

In some situations, viruses can infect other species, previously unknown to be susceptible to the infection, an event of cross-species transmission. This may be a rare finding, as it was the report of RHDV2 in Iberian hare in Spain [39], designated a spillover event, with no apparent consequences, or, on the contrary, may lead to severe infection in the new host. This is well exemplified by the emergence of a new recombinant myxoma virus (ha-MYXV) that acquired the capacity to infect Iberian hares, causing high mortality.

Identifying the viruses’ reservoirs provides crucial information for the knowledge of the epidemiology of the infections and the potential for transmission to other hosts. While MYXV reservoir is well known, RHDV2 reservoir is still unknown, despite some wild species, such as the Eurasian badger, are possible candidates.

2. Viral threats

Although the susceptibility of pikas to some viral agents such as Influenza [40] or Coronavirus [41] is recognized, to date no disease has been described that assumes great significance in terms of morbidity and mortality in this family, so they will not be described below.

2.1 Leporipoxviruses (MYXV, ha-MYXV and RFV)

2.1.1 Taxonomic classification

Leporipoxviruses affects mainly leporids and belong to Chordopoxvirinae subfamily, within Poxviridae family.

The following leporipoxviruses [42] and respective natural hosts are presently known:

1. Myxoma virus—Tapeti (Sylvilagus brasiliensis)

2. Californian myxoma virus—Brush rabbit (Sylvilagus bachmani)

3. Rabbit (Shope) fibroma virus—Eastern cottontail (Sylvilagus floridanus)

4. Squirrel fibroma virus—Eastern grey squirrel (Sciurus carolinensis)

5. Hare fibroma virus—European brown hare (Lepus europaeus)

6. Western squirrel fibroma virus—Western grey squirrel (Sciurus griseus griseus)

2.1.2 Morphology and genome organization

The MYXV strain Lausanne (Lu), Brazil/Campinas 1949, considered de facto the international reference strain (ATCC code VR-115), has a double-stranded DNA (dsDNA) genome with 161,777 bp of size and closed single-strand hairpin termini. The genome encodes a total of 158 ORFs with 12 duplicates in the 577 bp terminal inverted repeats (TIRs) [43, 44]. The viral genome is encapsidated in a brick-shaped virion, and the replication cycle occurs in the cytoplasm of infected cells where a spectrum of host-interactive immunomodulatory proteins is expressed [45].

Genes-encoding proteins involved in replication and structure are relatively conserved among other poxviruses and tend to be located in the central part of the genome, while genes at the termini of the genome tend to encode host-range and virulence factors [44, 46]. The function of 42 viral genes is related to the host range or immunomodulation [47].

2.1.3 Viral replication

The most well-studied intracellular replication cycle concerns vaccinia virus (VV). Both the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), which differ in their surface glycoproteins and in the number of layer membranes, can initiate the infectious cycle, using different mechanisms [48].

Poxvirus replication cycle begins with binding of the virus to the cell surface, probably ubiquitously expressed glycosaminoglycans or components of the extracellular matrix, triggering signalling events in several host protein-kinase cascades [48]. Subsequently, fusion of the virus envelope with the mammalian cell membranes occurs, with release of the virion core structure into the cytoplasm [48].

Once the virus core has been released into the cytoplasm, the cellular RNA polymerase and the encapsidated transcription factors initiate the first round of early viral gene expression, which synthesizes viral mRNA under the control of viral early promoters [49, 50]. Then, core uncoating releases the viral DNA into the cytoplasm, where it serves as template for DNA replication and for subsequent events of intermediate and late transcription, where host-derived transcription factors participate. As late viral gene products accumulate, progressive morphogenesis and assembly of infectious virus particles take place, initially as IMV virions, which assemble and migrate via microtubules being wrapped with Golgi-derived membranes to form intracellular enveloped virus (IEV) [48].

2.1.4 Epidemiology (origin, transmission and distribution)

Myxoma virus (MYXV)—reference virus, Rabbit fibroma virus (RFV) and more recently the natural recombinant myxoma virus (ha-MYXV or rec-MYXV) are the three poxviruses that most impact on leporids. Brazilian cottontail is the natural host of MYXV, while Eastern cottontail rabbit (S. floridanus) is the natural host of RFV, with both viruses causing benign and non-fatal infections in these hosts. On the contrary, in the European rabbit (O. cuniculus), RFV causes skin tumours (myxomas) with benign progression and MYXV causes impacting disease (named myxomatosis), generally lethal.

Until recently, MYXV infected only rabbits and European brown hares (L. europaeus), the last species only occasionally with clinical signs [51, 52, 53]. Apart from lagomorphs, MYXV is non-pathogenic for any of the hosts tested so far [52].

Myxomatosis was first described in 1896 in Montevideo, Uruguay, following the acquisition of European rabbit (O. cuniculus) for antiserum production [54]. The origin of the virus was attributed to the Eastern cottontail rabbit. After the disease being described, myxoma virus would come to be introduced by man in Chile (Tierra del Fuego), France and Australia [51, 55, 56] to control the extremely large rabbit populations, considered a plague, with excellent results in the short term but not effective in the medium and long term. The unsuccess of biological control happened again over time, either through the selection of less pathogenic virus strains or through the genetic selection of more resistant rabbits [42, 57, 58].

2.1.5 Pathogenesis and disease characterization

The replication of MYXV inoculated intradermally in European rabbit initiates in MHC-II+ cells at the dermal/epidermal interface. Then, the virus spreads to the draining lymph node, first replicating in cells of the subcapsular sinus and later in lymphocytes of T cell zones. Lymphocytes, and possibly monocytes, disseminate the virus to distal tissues, with low viral load detectable in the bloodstream [59, 60, 61]. Simultaneously, in the original inoculation site, the virus replicates in epidermal cells inducing hyperplasia and hypertrophy, disrupting the dermis and causing oedema and infiltration of mucoid material in the sub-dermis matrix—originating the myxoid tissue that is responsible for the virus’s name. The epidermal cells could also present ballooning degeneration with vesicle formation and disruption of dermis [62]. The visualization of cytoplasm eosinophilic inclusion bodies is possible but not common.

The higher virus titres are found in lymphoid tissues (>10⁸ pfu/g) with lymphoid depletion in the lymph nodes and, in some strains, in the spleen. High loads are also found in secondary cutaneous lesions, the swollen eyelids and particularly in the very swollen tissues at the base of the ears, which are probably important for insect transmission. The viral loads in the lungs or liver are generally lower [59, 60, 63].

This ability to replicate and disseminate via lymphocytes, establishing infection at distal sites, is critical to the virulence of the European rabbit [47, 64].

Virus shedding occurs by conjunctival and nasal secretions and by eroded cutaneous lesions. Secondary bacterial infections of the upper respiratory tract and conjunctiva with gram-negative bacteria (e.g., Pasteurella multocida and Bordetella bronchiseptica) are common, with infection of internal organs less reported [65]. When the duration of the disease is longer (i.e., in infections by less pathogenic strains), pneumonia is a common outcome [42, 66, 67].

MYXV strains can be grouped in five broad virulence grades based on the case fatality rates (CFR), average survival time (AST) and clinical signs [51, 68, 69], ranging from grade 1 to grade 5.

The pathophysiological process of death by myxomatosis is still poorly understood. The key viral proteins involved in the replication and dissemination in Sylvilagus species cause local immune suppression in the skin, allowing the virus to persist at this level. In the European rabbit, these proteins cause profound immunosuppression. The virulence of field strains was analysed by infecting small groups of laboratory rabbits, classifying them into five broad grades based on the case fatality rates (CFR), average survival time (AST) and clinical signs [51, 68, 69], namely:

1. Grade 1: CFR of 99.5%, AST ≤ 13 days

2. Grade 2: CFR of 95–99%, AST 14–16 days

3. Grade 3A: CFR of 90–95%, AST 17–22 days

4. Grade 3B: CFR of 70–90%, AST 23–29 days

5. Grade 4: CFR of 50–70%, AST 29–50 days

6. Grade 5: CFR less than 50%, AST not determined

Lepus granatensis was considered naturally resistant to myxomatosis, which is endemic in the Iberian Peninsula since 1956 [70]. In 2018, a naturally recombinant myxoma virus (named ha-MYXV or rec-MYXV) emerged in Iberian hare (L. granatensis) causing high mortality in the field, representing the first cases of non-sporadic/pathogenic myxomatosis in this species [71, 72]. This recombinant virus has more than 100 small mutations compared to the Lausanne strain, and an insertion of about 2.8 kb disrupting the M009.L gene, the most significant genetic difference, associated with the species barrier jump [72]. This same virus was later found associated with very virulent myxomatosis in domestic and wild rabbits, and the susceptibility of Oryctolagus cuniculus algirus to ha-MYXV strains isolated either from Iberian hare or from wild rabbit was also demonstrated [73, 74, 75, 76]. More recently, classic MYXV strains were found in Iberian hare (L. granatensis) and some cases in co-infection with recombinant MYXV strains [73, 76].

2.1.6 Clinical and laboratorial diagnosis

The clinical diagnosis of the disease is relatively easy although there are no pathognomonic signs and the clinical signs can be confused with other diseases such as Pasteurellosis. The lesions are mainly external and located on the skin, eyelids and genitalia, although respiratory pathology can also be found. The definitive diagnosis is obtained by laboratory techniques that have evolved over the years, namely through isolation in susceptible cell lines, electron microscopy, histopathology (which presents some very suggestive lesions, namely the presence of cytoplasmic inclusion bodies in the cells of the epidermis), among others and, above all, the use of the PCR technique, namely real-time PCR that allows a highly sensitive, specific and quantitative diagnosis in about 3 hours [77]. Recently, a multiplex PCR technique was developed allowing the diagnosis and differentiation the various strains of myxoma virus currently known to be circulating [78].

2.2 Lagovirus europaeus (RHDV (GI.1) and RHDV2 (GI.2))

2.2.1 Taxonomic classification

Rabbit haemorrhagic disease virus (RHDV) belongs to Lagovirus genus, one of the 11 genera that comprise the Caliciviridae family recognized by the International Committee on Taxonomy of Viruses (ICTV) [79]. Genus Lagovirus also includes European brown hare syndrome virus (EBHSV) and other non-pathogenic viruses, the rabbit caliciviruses (RCVs) or hare caliciviruses (HCVs) [80].

Rabbit haemorrhagic disease (RHD) can be caused by one of two distinct viruses, namely RHDV, also referred to as RHDVa, Lagovirus europaeus GI.1 or simply GI.1, and RHDV2, also known as RHDVb, Lagovirus europaeus GI.2 or simply GI.2.

2.2.2 Morphology, genome organization and phylogenetic data

The aetiological agent of rabbit haemorrhagic disease (RHD) is a single-stranded, non-enveloped icosahedral capsid virus with a spherical morphology and a positive-sense RNA [81, 82, 83].

The major capsid protein VP1/VP60 forms the structure of the virion and the minor structural protein VP2/VP10 is responsible for stability after the encapsidation of viral RNA, covalently linked to the VPg (viral protein genome-linked), which is essential for replication [80].

The genome is around 7.4 kb (precisely 7437 nucleotides long) to which the 2.2 kb subgenomic RNA (sgRNA) binds to. The genomic RNA is divided into ORF1, encoding a polyprotein that is cleaved into several non-structural proteins and the major structural capsid protein, VP60 (60 kDa), and ORF2 that encodes the minor structural protein called VP10 or VP2 [81, 82, 84].

The viral capsid comprises 90 arch-like dimers of the capsid protein VP60, and 32 cup-shaped depressions, which gives the name to the family Caliciviridae, arranged in a T = 3 icosahedral symmetry [85, 86, 87, 88]. The electron-dense core has an approximate diameter of 23–25 nm [89, 90].

In the VP60 the main viral antigen, the exposed surface loop—L1 from the P2 subdomain exhibits a higher variability between the strains and contains neutralizing antibody inducing epitopes [91, 92, 93]. The cross-protective immunity between the different genotypes is very limited [91, 92, 93, 94, 95, 96] and for this reasons, it is predictable that new RHDV genogroups will keep emerging in the future.

2.2.3 Viral replication

The suggested primary site of RHDV replication and entry door (probably by binding to ABH histo-blood group antigens (HBGAs)) is the epithelial cell of the upper respiratory and digestive tracts, being the hepatocyte the major site of replication [97, 98]. The viral genome is released into the cell cytoplasm leading to the direct translation of the viral proteins. In the absence of m7G cap strucures, VPg may play a crucial role in the translation initiation [99, 100] acting as a cap substitute or analogue, interacting with translation initiation factors eIF4E and/or eIF3 [100, 101].

While the translation of ORF1 encoding polyprotein precursor occurs at the initiation codon AUG, the translation of ORF2 encoding VP10 starts by an unusual mechanism of reinitiation after the termination of translation of the preceding major capsid protein VP60 [99]. VP10 can induce hepatocyte apoptosis and virion release and dissemination [102].

2.2.4 Epidemiology (origin, transmission and distribution)

RHDV (Lagovirus europaeus GI.1) emerged in Wuxi City, in the last quarter of 1983 in domestic rabbits imported from Germany to the Jiangsu province in China [83, 103], being the first genotype described of rabbit haemorrhagic disease virus (RHDV) [103, 104], that causes a fatal disease in adult rabbits and a subclinical disease in rabbits younger than 4–6 weeks [105, 106]. In the first 12 months after its emergence, the disease killed over 140 million rabbits in China and reached the Europe, namely to Italy, 2 years later [107, 108]. Within 10 years, the disease became endemic in Europe, with severe impact on the European wild rabbit, mainly on the Iberian Peninsula where the specie is a keystone species [14, 23, 109], but also causing sever loses in industrial rabbit farms in both Europe and North Africa [110].

The first cases of RHDV in wild rabbits were reported in Spain in 1988 [111], in Madeira Island (in 1988) and in the Azorean archipelago (Faial Island in 1988, São Jorge Island in 1989 and Santa Maria Island in 1990) (reviewed in Ref. [112]). In the next years, the disease was reported worldwide.

RHDV2 emerged in France in 2010 [113] and quickly replaced the circulating strains of RHDV in most European countries, in the both wild and domestic populations [114, 115, 116]. Nowadays, the virus is already reported almost all over the world [112, 113, 117, 118, 119, 120, 121, 122, 123, 124].

In the last decade, RHDV2 was reported in several non-habitual species, some representing species barrier jumps including for different mammalian orders, such as small mammals species [96] as well as in Alpine Musk Deer (Moschus sifanicus) [125] and Euroasiatic badger (Melus melus) [126].

2.2.5 Pathogenesis and disease characterization

RHDV is the etiological agent of the Rabbit Haemorrhagic Disease (RHD), the so-called given the severe dysregulation of the coagulation system.

RHDV integrates the WOAH list of notifiable terrestrial and aquatic animal diseases being a transmissible disease of socio-economic importance within countries, and significant in the international trade of animals and animal products.

The incubation period of RHD induced by RHDV (GI.1) ranges from 1 to 3 days [111, 127] while for RHDV2 (GI.2), it ranges between 3 and 9 days with death occurring 12–36 hours after the onset of fever.

RHDV2 also differs from RHDV in antigenic profile, the apparent lower mortality (5–70%, 20% in average), compared to RHDV [106, 113, 128], age of the animals affected (RHDV2 affects kittens of just 11 days [117]), a longer course of disease of 3–5 days and a higher proportion of rabbits showing subacute-chronic disease comparing with the previous genotypes [106, 113]. The virus has already been detected in leporids other than the European rabbit, namely in the L. europaeus [129], L. capensis [130], L. timidus [131, 132], L. granatensis [39], L. californicus and Sylvilagus audubonii [123].

2.2.6 Clinical and laboratorial diagnosis

The typical (nodular) myxomatosis shows very specific, but not pathognomonic, lesions, particularly nodular thickening of the eyelids, the presence of myxomas and anogenital oedema. The final diagnosis may be achieved by molecular diagnosis according to the WOAH guidelines [133].

Poxviruses can be identified in the skin lesions (myxomas), eyelids and genitalia but also in many other organs attending to the fact that the disease is most often systemic, so in the lungs, liver, spleen and kidney, among others [133]. The diagnosis can be performed through laborious techniques such as the negative-staining electron microscopy (nsEM), histopathology, immunohistochemistry, viral isolation, agar gel immunodiffusion (AGID) and direct immunofluorescence test (dFT) but is currently mostly performed using real-time PCR [78].

2.3 European Brown Hare Syndrome Virus (EBHSV)

2.3.1 Taxonomic classification

Like RHDV, European Brown Hare syndrome Virus (EBHSV) belongs to the Lagovirus genus and the Caliciviridae family. Although not recognized by the ICTV, the proposed classification [104] for this virus is Lagovirus europaeus (species), which includes genogroup GII (EBHSV and hare calicivirus (HaCV)). EBHSV represents the GII.1 genotype that includes three variants (GII.1a, GII.1b and GII.1c).

EBHSV shares with RHDV a phylogenetic relationship, however with distinct antigenic profiles [89, 134].

2.3.2 Epidemiology (origin, transmission and distribution)

The disease was first reported in Gotland island, Sweden, in 1980 and, 1 year later, in the mainland [135]. In the following years, the disease spread to several European countries [136, 137, 138, 139] with a huge impact on the wild populations of European Brown hare [140, 141, 142, 143].

The European Brown Hare Syndrome (EBHS) shares with RHD many pathophysiological, clinical and epizootic particularities. This disease is also an highly infecto-contagious and fatal disease of the European brown hare (Lepus europeaus), the most disseminated hare species, and in lower grade the Mountain hare (Lepus timidus) found in the tundra biome [144].

Generally, hares develop an acute form of disease dying a few hours after clinical signals onset. The young hares are also susceptible to infection but due to eventual natural resistance, do not develop disease [89, 144].

EBHSV may be less species-specific than RHD virus and has been recorded as infecting also Eastern cottontails (S. floridanus).

2.3.3 Clinical and laboratorial diagnosis

Death is most often the outcome of acute infections, so free-ranging hares may be found dead in the field if not predated. The clinical course is very similar to RHD, with anorexia, depression and neurologic signs secondary to hepatic encephalopathy.

Gross and microscopic lesions may include hepatocellular necrosis, haemorrhages, icterus, inflammatory infiltrates, splenic and/or renal congestion and enlargement [145].

The confirmation of diagnosis may be carried out by molecular techniques namely conventional or real-time PCR as well as using indirect methods that detect antibodies [145].

2.4 Leporid herpesvirus (LeHV-1 to LeHV-5)

2.4.1 Taxonomic classification

Herpesviruses affecting mammals, birds and reptiles belong to Herpesviridae family and are highly disseminated in nature, with most animal species have yielded at least one herpesvirus identified [146].

The Herpesviridae family includes the subfamilies Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae. Their members have different biologic properties and distinct classification, supported by phylogenetic data. The Gammaherpesvirinae subfamily is divided into four genera, namely Macavirus, Percavirus, Lymphocryptovirus and Rhadinovirus [147, 148].

While members of the subfamily Alphaherpesvirinae cause rapid lysis in cell culture, members of Betaherpesvirinae grow slowly inducing the formation of giant cells in culture, while Gammaherpesvirinae typically infect lymphoid tissue, revealing a primary tropism for lymphoid lineage cells [149], which can lead to lymphoproliferative diseases [150] and oncogenesis [151].

At the moment, five herpesviruses were described in Leporids namely the Leporid herpesvirus 1 (LeHV-1), Leporid herpesvirus 2 (LeHV-2), Leporid herpesvirus 3 (LeHV-3), Leporid herpesvirus 4 (LeHV-4) and Leporid herpesvirus 5 (LeHV-5). LeHV-2 and LeHV-3 (reviewed by Refs. [150, 152]) are the most commonly described herpesviruses in rabbits, which alongside LeHV-1 belong to the Gammaherpesvirinae subfamily. LeHV-4, an alphaherpesvirus, is the most pathogenic in rabbits, causing fatal infections. Recently, LeHV-5 (Leporid gammaherpesvirus 5) was described affecting the Iberian hare [153].

2.4.2 Morphology and genome organization

The virion structure [146] consists of a core containing a linear dsDNA with 124–295 kb in length, an icosahedral capsid with approximately 125 nm in diameter, one capsomeric structure that serves as the portal for packaging and release of the viral genome, the tegument—an amorphous appearing substance that surrounds the nucleocapsid, and an envelope containing viral glycoprotein spikes on its surface. When mature, the virion size ranges from 120 to 260 nm [154]. The envelope derives from patches of altered nuclear membrane being mainly constituted by viral glycoproteins [155, 156].

The core of a mature virion contains a single dsDNA packed in a torus form [155, 157]. The capsid contains 161 capsomers (10 hexons and 11 pentons), a portal complex with a capsid triangulation (T = 16), preserved in all herpesviruses [158, 159, 160]. The non-enveloped capsids could present different forms, namely A (capsids without core), B (capsids containing the assembly scaffold without genome) and C (capsids containing genome without scaffold) [161].

The tegument is a proteinaceous structure between the nucleocapsid and the envelope [146] in general thicker in the virions accumulating in cytoplasmic vacuoles. The tegument proteins closer to the nucleocapsid (inner tegument) are acquired in the nucleus and by interactions with envelope glycoproteins [162] and the subsequent components incorporated in the cytoplasm [162]. The tegument proteins are important in the early phase of infection. The envelope derives from patches of altered nuclear membrane being mainly constituted by viral glycoproteins [155, 156].

The genome of Herpesviridae members encodes between 70 (the smallest) and 200 (the largest) proteins [146].

Herpesvirus genomes can be divided into six groups designated [146] as follows:

1. Exemplified by Human herpesvirus 6 (HHV6), where a large sequence from one terminus is directly repeated at the other terminus;

2. Exemplified by herpesvirus saimiri (SaHV-2), where the terminal sequence is directly repeated numerous times at both termini, with a variable number of repeats at the termini;

3. Exemplified by Epstein-Barr virus (EBV), where the number of direct terminal repeats is smaller and can harbour other direct sequence arrays that subdivide the unique (or quasi unique) sequences of the genome into several well-delineated stretches;

4. Exemplified by Varicella-Zoster virus (VZV), where one terminus is repeated in an inverted orientation internally. The domain consisting of the stretch of unique sequences flanked by inverted repeats (Small or S component) can invert relative to the remaining sequences (Large or L component);

5. Exemplified by herpes simplex virus (HSV) and human cytomegalovirus (HCMV), where the sequences from both termini are repeated in an inverted orientation and juxtaposed internally, dividing the genomes into two components, each of which consists of unique sequences flanked by unrelated pairs of inverted repeats;

6. Exemplified by tupaia herpesvirus 1 (TuHV-1), where the terminal sequences are not identical and are not repeated either directly or in an inverted orientation.

The replication cycle occurs in three major phases: initiation of infection, lytic replication and latency [146]. A biological decision at the cell level is taken to follow either the lytic or the latent pathway, as well, after the initial infection and latency, reactivation of a lytic state [146].

During the lytic replication, a cascade of lytic gene expression occurs, management of the host cell, management of adaptive immune response, replication of virus genome, virus assembly and egress and transmission to other cells and hosts [146]. The restriction of lytic gene expression and expression of latency genes that manage the cell and host defences and maintain the virus genome in the infected cells leads to the latent pathway [146].

2.4.3 Viral replication

The replication cycle is divided into three main phases: initiation of infection, lytic replication and latency [146]. During the initial phase, the binding to the cell receptor occurs, followed by fusion of the viral membrane with the plasma membrane or after endocytosis, management of intrinsic response by tegument proteins, transport of nucleocapsid and tegument-associated IE-activators to the nucleus, injection of viral genome through the nuclear pores and genome chromatinization. The initial interactions with the transcriptional machinery than take place [146]. After these initials steps, the infection triggers a lytic or a latent pathway and in the case of latency, reactivation may occur under certain conditions [146]. During the latent pathway, occurs restriction of lytic gene expression and expression of latency genes that manage the cell and host defences [146].

2.4.4 Epidemiology (origin, transmission and distribution)

The LeHV-5 described in 2020, the only herpesvirus described in hares so far, has been shown to have a great impact on the morbidity and mortality of the Iberian hare, especially when associated with ha-MYXV infections [153]. A survey carried out in Portugal mainland between 2018 and 2021 (Project +Coelho and Project +Coelho 2) showed that approximately 29% of the hares tested (n = 101) were positive for LeHV-5 and all of these were simultaneously coinfected with ha-MYXV.

2.4.5 Pathogenesis and disease characterization

Grossly, vesiculopustular lesions on the eyelids, snout, lips and genitals, necrotizing balanoposthitis and necrosuppurative inflammatory processes on the eyelid and the perivulvar region were observed. Histopathological analysis showed typical herpetic-like vesicles in the epidermis and in the stroma, and a proliferation of pleomorphic spindle cells in the dermis, with nuclei displaying slightly eosinophilic inclusion bodies (Cowdry type A inclusions). The co-infection between LeHV-5 and ha-MYXV leads to an aggressive clinical course and almost invariably to death.

A review of the different herpesviruses and their pathogenies was recently published [153]. Since 2010 no cases of herpesvirus have been reported in both domestic and wild rabbits, and the known herpesviruses have been described very few times over the years.

3. Prevention and disease control of wild leporid viral infections

MYXV grows well in cell lines and live-attenuated viruses through cell passages have been extensively used in Europe as vaccines, due to the inefficacy of inactivated vaccines. On the contrary, until recently, only inactivated vaccines were available [163, 164, 165, 166, 167, 168] since RHDV/RHDV2 cannot be grown in cell culture.

The first vaccine available for myxomatosis was an heterologous rabbit fibroma virus [169]. A decade later, an homologous vaccine offering longer immunity was produced based on strains such as MYXV MSD strain, French SG33, MAV, among others, by attenuation in cell culture [170]. Nowadays, these vaccines are still being used and MYXV is also used as a vector for recombinant vaccines harbouring the VP60 of Lagovirus europaeus.

Despite the good efficacy of commercial vaccines against myxomatosis, there are still sporadic outbreaks in industrial rabbit farming, with annual rabbit deaths and costs of biosecurity measures [171]. The outbreaks in industrial rabbit farming often result from vaccine failures not associated with the vaccine itself, but rather with the route of administration, as it has been demonstrated that the intradermal route triggers a better seroconversion than other routes, namely the subcutaneous route, the most commonly used [172]. This fact exemplifies the need and benefits of bringing the productive sector closer to the scientific researchers in order to standardize more effective methods of administration and control of this and other diseases. In fact, the recently emerging ha-MYXV, which has been shown to infect wild and domestic rabbits as well, is highly pathogenic, but commercial vaccine strains have been shown to be effective in preventing disease in rabbits [73, 74, 75].

On the other hand, the low applicability of vaccination in the wild hampers a practical solution to contain the virus, resulting in the continuous source of infection and circulation of the virus. Myxomatosis has had a major impact on wild rabbit and Iberian hare populations since its emergence leading to abrupt reductions and local extinctions [3, 173, 174, 175, 176] with a chain effect, especially in the Mediterranean ecosystems, due to the role of keystone species in food chains [5].

Myxomatosis treatment is possible, but merely symptomatic, considering that there are no antivirals tested for this disease, being a utopia in the case of wild species due to the difficulty in capture and administration. Moreover, treatment is not recommended, due to the risk of environmental contamination and transmission potentiation during treatment (taking into account the dispersion by biting insects and mechanical transmission), and the possibility of the animals becoming carriers [177]. For this reason, the only current way of controlling the disease is through vaccination and biosecurity measures, which can be adapted, partially, to the wild.

Since 2018, thousands of hares died in Portugal and Spain in the field due to ha-MYXV, causing a reduction of more than 50% in the wild populations of both countries, which will lead to the attribution of Vulnerable status by the IUCN in late 2022. The long-term impact of this new recombinant virus on the Iberian hare and the wild rabbit is yet to be known. However, the possibilities available for the recovery of both species are predictably low, especially for the Iberian hare, which has a dynamic reproduction much lower than the wild rabbit.

4. Conclusions

In recent years, the wild rabbit and the Iberian hare have been gaining growing interest from the academy, civil society, the environmental and ecological organizations and policy makers. A clear example of these was the Projects +Coelho, implemented following the constitution of a working group by Dispatch no. 4757/2017 of 31 May of the Portuguese Ministry of Agriculture, to respond to the effect of rabbit haemorrhagic disease in the rabbit population, the Mixolepus project in Spain in response to the emergence of ha-MYXV or the recently approved LIFE Iberconejo with an allocation of around 2 million euros for 3 years. These, and other investments, are fully justified given the importance of the wild rabbit in the Mediterranean ecosystem, where its role is so decisive that some ecologists call the Iberian Peninsula the “rabbit’s ecosystem” [5]. In fact, the most paradigmatic conservation projects in the Iberian Peninsula take place on areas with rabbit abundance namely the projects involving Iberian lynx (Life+IBERLINCE), Black vulture (Parque Natural do Tejo Internacional) and Imperial eagle (Parque Natural do Vale do Guadiana and ZPE of Castro Verde), among others.

Viral diseases have been identified as the main causes of the leporid decline [115, 116]. The importance and severity of viruses were evidenced during the 3 years of field work performed within the scope of this doctoral thesis when it was possible to testify the emergence of myxomatosis in Iberian hare by the natural recombinant ha-MYXV [71], cases of co-infection with ha-MYXV and classic MYXV in hares and rabbits, never detected before [76], co-infection with MYXV and RHDV2 [178] the spillover of the ha-MYXV (natural recombinant myxoma virus) from hares to rabbits [74, 75], the identification of a herpesvirus in hares undergoing myxomatosis [74, 75] not described before and the detection of an Iberian hare infected with rabbit haemorrhagic disease [39]. During the health analysis of wild rabbit cadavers found in mainland Portugal between 2018 and 2020, MYXV was found in about 27.81% of the animals [3]. Also in the scope of the virological analyses of leporid cadavers found in mainland Portugal between 2018 and 2020, RHDV2 was found in about 48.52% of the rabbits and 84.21% of the hares [3]. During the health analysis of rabbit cadavers found in mainland Portugal between 2018 and 2020, RHDV2 and MYXV were detected in 76.33% [116] and associated with animal death.

5. Future perspectives

Currently, only the viral infections described above are associated with a major impact on wild leporid populations, with particular relevance to myxoma viruses and Lagoviruses. However, because only what is sought is found, we cannot guarantee that other viruses are not affecting the wild populations. In fact, beside a few time-limited studies, no robust and long-term sanitary surveillances and research programmes have been conducted in wild leporid species. It is therefore crucial to invest in continuous monitoring of these species, that are essential for ecosystems, with a special focus on the Mediterranean basin.

The Iberian hare is an endemic specie of the Iberian Peninsula whose populations are considered stable by the IUCN holding a ‘least concern’ conservation status [179]. However, its status will be likely reviewed this year, probably to Vulnerable, given the trends registered after 2018. If the current downward trend in the field continues, and if myxomatosis also becomes endemic in this species, the L. granatensis conservation status will be progressively worsened to the point of functional extinction of a species that is iconic for the Iberian Peninsula.

The progressive loss, fragmentation and changes in habitat and wildlife management are putting leporids, and other species around the world, at great risk. Only a professional and integrated management of these species will allow them to remain as key species in our ecosystems. Otherwise we will continue to observe the drastic cascading effects of leporid decrease on other species.

According to the World Wildlife Fund (WWF) ‘Living Planet 2020’ report, global wildlife populations have declined by an average of 60% over the past 40 years; therefore, it is urgent to adopt practical measures of positive impact in short and medium terms [180].

Today, we have the knowledge and the means to develop solutions, often retained by lack of political will and social and ecological motivation, to make pioneering and innovative decisions that will allow paradigm shifts. One such example is the use of new microbiological solutions, namely recombinant vaccines, transgenic foods that express immunizing proteins, the use of artificial intelligence and machine learning to deliver molecules. All these resources, and many more, are now a reality, but they still need to be implemented in wildlife.

Acknowledgments

The authors thank the Virology laboratory technical staff of the National Institute for Agricultural and Veterinary Research (INIAV, I.P.). Some data reported here resulted from funding by Fundo Florestal Permanente (Government of Portugal) within the scope of the Action Plan for the Control of Rabbit Viral Haemorrhagic Disease (Project +COELHO, Dispatch no. 4757/2017 of 31 May), Fundação para a Ciência e Tecnologia (FCT, UIDB/00276/2020), Lisbon, Portugal and the Interdisciplinary Research Centre on Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon, Portugal.

Conflict of interest

The authors declare no conflict of interest.