Summary
The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) , has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported.
To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares (88 live captured, 313 hunted and 50 found dead) collected over two time periods, 1994-1999 and 2017-2019, using a rabbit commercial indirect ELISA after validation, and tested different tissues or sera by a qPCR targetingM0005L/R gene conserved in MYXV and ha-MYXV.
The cut-off of ELISA Relative Index 10 = 6.1 yielded an estimated positive predictive value of 96.4% (CI95%82.6-98.0%), by comparison with qPCR positive and negative reference hares. Overall, antibodies were detected in 12.6% (57/451) of the hares tested, of which 40.3% (23/57) were also qPCR positive. Antibodies were found in apparently healthy hares sampled in 1994-1999 (n=10, none MYXV-DNA positive), and in 2017-2019 (n=28, of which 14% were MYXV-DNA positive). For the Iberian hares hunted or live trapped, seroprevalence was significantly higher in 2017-2019 (13.0%, CI95%9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p=0.005), and significantly higher in 2019 (p=0.007), being lower during the winter (p<0.001).
While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1994, they also show an increase in seroprevalence in 2018-2019. The more remote contact of hares with MYXV may have occurred with strains that circulated in wild rabbit, or unnoticed strains circulating in Iberian hare populations. This work clearly confirms the circulation of MYXV in the Iberian hare ate least 20 years before the severe virus outbreaks observed in 2018.
Keywords: emerging disease, Lepus granatensis , Iberian hare, myxomatosis, myxoma virus, recombinant, qPCR, ELISA, Iberian Peninsula
Introduction
Myxomatosis is caused by infection with the myxoma virus (MYXV), belonging to genus Leporipoxvirus from subfamily Chordopoxvirinae and family Poxviridae. It has a 163 kbp-long dsDNA genome, that/which replicates in the cytoplasm of the infected cells (Murphy et al., 1995), and is transmitted mainly by biting arthropods or direct contact with infected animals (Mead-Briggs and Vaughan, 1975).
In the European rabbit (Oryctolagus cuniculus ), MYXV high virulent strains induce a severe, often fatal, generalised disease characterised by swollen head, eyelids and ears, blepharoconjunctivitis with mucopurulent ocular and nasal discharge, cutaneous lesions, and inflammation and oedema of the genitalia and perianal skin (Bertagnoli and Marchandeau, 2015; Kerr et al., 2015). Since its intentional introduction in Europe in 1952, myxomatosis became endemic in European rabbit populations (Bertagnoli and Marchandeau, 2015; Villafuerte et al., 2017a). According to the severity of the lesions induced in rabbit, the MYXV strains can be classified in I (higher) to V (lower) virulence grades ( Kerr & Best, 1998).
Until recently, the European rabbit was the only Iberian lagomorph considered highly susceptible to infection by the MYXV. While sporadic cases of myxomatosis have been reported in brown hares Lepus europaeus (Magallon et al., 1953; Jacotot et al., 1954; Barlow et al., 2014), the endemic and declining Iberian hare (Lepus granatensis ) (Carro and Soriguer, 2017) was considered resilient to MYXV infection, with no confirmed cases of myxomatosis until 2018, when a large outbreak was reported in southwestern Spain (Bocanegra et al., 2019). This outbreak recurred on the subsequent years, and has been spreading throughout the Iberian Peninsula (Carvalho et al., 2020). Between September 2017 and mid-October 2018, within a national Portuguese surveillance program, 80 asymptomatic hunted Iberian hares were investigated for MYXV-DNA, with none testing positive (Duarte, M.D. et al., 2018).
A novel strain of MYXV was genotyped from dead Iberian hares during the 2018 outbreak (Dalton et al., 2019; Pinto et al., 2019; Carvalho et al., 2020).These studies demonstrated a natural recombinant virus (ha-MYXV), which has additional genetic material, hypothesized to have allowed the species jump event, and/or increased virulence for Iberian hares. Although ha-MYXV DNA was detected in few wild rabbits (unpublished data ), the rarity of these findings supports the apparent preferential circulation of MYXV and ha-MYXV in rabbits and hares, respectively.
The susceptibility of the Iberian hare to MYXV infection (sensu latu ) has only recently been reported, associated with a recombinant virus never described before. However, neither the susceptibility of the hare to MYXV infection, nor the lack of it, has been scientifically investigated.
The aim of this study is to provide insights into the emergence of myxomatosis in the Iberian hare, by assessing the presence of antibodies against MYXV antigens in specimens from Spain and Portugal, before and during the outbreaks reported in 2018.
We performed a retrospective serological survey in samples collected in two periods different periods, with 18 years apart; before the outbreak (1994 to 1999) to investigate the contact of Iberian hare populations with MYXV or MYXV-like viruses in the past, and encompassing the outbreak recorded in 2018. Whenever possible, the serological data was combined with clinical, pathological and molecular information to explore uncovered clues about the recent emergence of highly pathogenic ha-MYXV in Iberian hares.
Given that the origin of this recombinant virus is yet to be explained, several hypotheses are revised and considered in this manuscript.