Myxoma virus (MYXV) and rabbit haemorrhagic disease virus 2 (RHDV2) are two major pathogens that affect the European rabbit ( Oryctolagus cuniculus ). Between August 2017 and August 2019, 1166 wild rabbits (971 legally hunted and 195 found dead) were tested by PCR‐based methods for MYXV and RHDV2 within the scope of an ongoing surveillance programme on wild leporids in Portugal. Despite never having been reported before and being considered a rare event, coinfection by RHDV2 and MYXV was detected in one juvenile wild rabbit found dead in the Évora district located in Alentejo. The relative frequency of coinfection in the group of diseased rabbits (found dead in the field) was 0.52 per cent (1/195). The positivity percentage of each single virus was much higher, namely, 14.36 per cent (28/195) for MYXV and 55.38 per cent (108/195) for RHDV2, within the 2 years of sample collection considered.

Rabbit hemorrhagic disease virus 2/genotype GI.2 (RHDV2/GI.2; Caliciviridae, Lagovirus) causes a highly contagious disease with hepatic necrosis and disseminated intravascular coagulation in several Leporidae species. RHDV2 was first detected in European rabbits ( Oryctolagus cuniculus) in France in 2010 and has since spread widely. We gather here data on viral detections reported in various countries and affected species, and discuss pathology, genetic differences, and novel diagnostic aspects. RHDV2 has been detected almost globally, with cases reported in Europe, Africa, Oceania, Asia, and North America as of 2023. Since 2020, large scale outbreaks have occurred in the United States and Mexico and, at the same time, cases have been reported for the first time in previously unaffected countries, such as China, Japan, Singapore, and South Africa, among others. Detections have been notified in domestic and wild European rabbits, hares and jackrabbits ( Lepus spp.), several species of cottontail and brush rabbits ( Sylvilagus spp.), pygmy rabbits ( Brachylagus idahoensis), and red rock rabbits ( Pronolagus spp.). RHDV2 has also been detected in a few non-lagomorph species. Detection of RHDV2 causing RHD in Sylvilagus spp. and Leporidae species other than those in the genera Oryctolagus and Lepus is very novel. The global spread of this fast-evolving RNA virus into previously unexploited geographic areas increases the likelihood of host range expansion as new species are exposed; animals may also be infected by nonpathogenic caliciviruses that are disseminated by almost all species, and with which genetic recombination may occur.

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 collected over two time periods almost two decades apart, 1994–1999 and 2017–2019 for the presence of antibodies and MYXV‐DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha‐MYXV. The cut‐off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related‐antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994–1999, none MYXV‐DNA positive, and n = 28 sampled in 2017–2019 of which four were also ha‐MYXV‐DNA positive) and 19 found‐dead and ha‐MYXV‐DNA‐positive sampled in 2018–2019. Interestingly, four seronegative hares sampled in 1997 were MYXV‐DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), 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 = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .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 1996, they also show an increase in seroprevalence in 2018–2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

During the necropsies of Iberian hares obtained in 2018/2019, along with signs of the nodular form of myxomatosis, other unexpected external lesions were also observed. Histopathology revealed nuclear inclusion bodies in stromal cells suggesting the additional presence of a nuclear replicating virus. Transmission electron microscopy further demonstrated the presence of herpesvirus particles in the tissues of affected hares. We confirmed the presence of herpesvirus in 13 MYXV-positive hares by PCR and sequencing analysis. Herpesvirus-DNA was also detected in seven healthy hares, suggesting its asymptomatic circulation. Phylogenetic analysis based on concatenated partial sequences of DNA polymerase gene and glycoprotein B gene enabled greater resolution than analysing the sequences individually. The hare’ virus was classified close to herpesviruses from rodents within the Rhadinovirus genus of the gammaherpesvirus subfamily. We propose to name this new virus Leporid gammaherpesvirus 5 (LeHV-5), according to the International Committee on Taxonomy of Viruses standards. The impact of herpesvirus infection on the reproduction and mortality of the Iberian hare is yet unknown but may aggravate the decline of wild populations caused by the recently emerged natural recombinant myxoma virus.