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.