Rabbit Viral Haemorrhagic Disease control in Iberian ecosystems: dynamics of virus spread, pathophysiology and improvement of prophylactic measures.

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Impact of leporid viral diseases in Iberian ecosystems : emergence, pathophysiology, prophylaxis and diagnosis

Publication . Santos, Fábio Alexandre Abade dos; Duarte, Margarida Dias; Peleteiro, Maria da Conceição Cunha e Vasconcelos; Parra Fernández, Francisco

ABSTRACT - The alarming state of conservation of leporid species in the Iberian Peninsula justifies the adoption in this Doctoral Thesis of an integrative approach that includes the study of different aspects of the virus-host-environment interfaces with respect to the main viral pathogens of the wild rabbit (Oryctolagus cuniculus algirus) and the Iberian hare (Lepus granatensis) in Mediterranean ecosystems. The studies to which this thesis refers to have used and developed methodologies of molecular and cellular biology, genetics, virology, immunology and pathology, applied to the wild rabbit and the Iberian hare, establishing as objectives of this Thesis the pathophysiological research of the diseases caused by rabbit hemorrhagic disease virus (RHDV2), myxoma virus (MYXV) and leporid gammaherpesvirus 5 (LeHV-5), their impact on affected species, the design of new diagnostic methods and the search for control measures to mitigate the effects of these agents. In addition to reflecting on the correct interpretation of molecular diagnoses in the context of the virus-host relationship, as a previous step to the presentation of the experimental results, some methodologies have been developed which were necessary for their implementation, such as the extraction of blood through the external jugular vein in wild rabbits and Iberian hares, or simple procedures in order to obtain primary cultures from leporid fibroblasts. In this thesis it has been shown that the Eurasian badger (Meles meles) is susceptible to RHDV2 and that this species can act as a potential reservoir of the rabbit hemorrhagic disease virus. It was also shown that the same RHDV2 strain isolated from badgers could infect a dwarf rabbit, who had a prolonged (atypical) illness and a history of vaccination ineffectiveness with a commercial RHDV vaccine still available on the Portuguese market. With regard to myxomatosis, this thesis includes the detection and analysis of the first cases of this disease in Iberian hares, associated with a natural recombinant strain of the virus (ha-MYXV), as well as the first demonstration that the recombinant virus of hares can infect both wild and domestic rabbits. Additionally, evidence of the first cases of co-infection of the recombinant virus ha-MYXV and the classic myxoma virus MYXV is reported, both in wild rabbits and in Iberian hares. In an attempt to provide solutions for the control of myxomatosis in wild leporids, it has been shown that commercial homologous vaccines against myxomatosis are effective in wild rabbits against the new naturally recombinant strain ha-MYXV, but failed to protect the Iberian hare against myxomatosis. ...

2022-12-05Doctoral thesis

Open access

Spillover events of rabbit haemorrhagic disease virus 2 (recombinant GI.4P-GI.2) from Lagomorpha to Eurasian badger

Publication . Abade dos Santos, F.A.; Pinto, Andreia; Burgoyne, Thomas; Dalton, Kevin P.; Carvalho, Carina L.; Ramilo, David; Carneiro, Carla; Carvalho, Tânia; Peleteiro, Conceição; Parra, Francisco; M. Duarte, M. D. Duarte, M. M. Duarte

Rabbit haemorrhagic disease (RHD) is a major threat to domestic and wild European rabbits. Presently, in Europe, the disease is caused mainly by Rabbit haemorrhagic disease virus 2 (RHDV2/b or Lagovirus europaeus GI.2), the origin of which is still unclear, as no RHDV2 reservoir hosts were identified. After the RHDV2 emergence in 2010, viral RNA was detected in a few rodent species. Furthermore, RHDV2 was found to cause disease in some hare species resembling the disease in rabbits, evidencing the ability of the virus to cross the species barrier. In this study, through molecular, histopathologic, antigenic and morphological evidences, we demonstrate the presence and replication of RHDV2 in Eurasian badgers (Meles meles) found dead in the district of Santarém, Portugal, between March 2017 and January 2020. In these animals, we further classify the RHDV2 as a Lagovirus europaeus recombinant GI.4P-GI.2. Our results indicate that Meles meles is susceptible to RHDV2, developing systemic infection, and excreting the virus in the faeces. Given the high viral loads seen in several organs and matrices, we believe that transmission to the wild rabbit is likely. Furthermore, transmission electron microscopy data show the presence of calicivirus compatible virions in the nucleus of hepatocytes, which constitutes a paradigm shift for caliciviruses’ replication cycle

2022-05Journal article

Open access

Evaluation of commercial myxomatosis vaccines against recombinant myxoma virus (ha-MYXV) in Iberian hare and wild rabbit

Publication . Abade Dos Santos, F.A.; Carvalho, Carina; Valente, Pamela; Armes, Henrique; Reemers, Sylvia S.; Peleteiro, Maria C.; Calonge Sanz, Ines; Dalton, Kevin P.; Parra, Francisco; M. Duarte, M. D. Duarte, M. M. Duarte

The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with the Mixohipra-H vaccine or the Nobivac Myxo-RHD PLUS (MSD Animal Health) using the recommended doses for domestic rabbits. After the vaccination trials, the animals were challenged with a wild type strain of ha-MYXV. The results showed that no protection to ha-MYXV challenge was afforded when a commercial dose of Mixohipra-FSA or Mixohipra-H vaccine was used in hares. However, the application of a higher dose of Mixohipra-FSA vaccine may induce protection and could possibly be used to counteract the accelerated decrease of wild hare populations due to ha-MYXV emergence. The two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) tested in wild rabbits were fully protective against ha-MYXV infection. This knowledge gives more insights into ha-MYXV management in hares and rabbits and emphasises the importance of developing a vaccine capable of protecting wild populations of Iberian hare and wild rabbit towards MYXV and ha-MYXV strains

2022-02-24Journal article

Open access

A versatile qPCR for diagnosis of leporid gammaherpesvirus 5 using Evagreen® or Taqman® Technologies

Publication . Abade Dos Santos, F.A.; Carvalho, Carina L.; Peleteiro, Maria C.; Parra, Francisco; Duarte, M. D.

ABSTRACT - In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.

2021-04-20Journal article

Open access

Co‐infection by classic MYXV and ha‐MYXV in Iberian hare (Lepus granatensis) and European wild rabbit (Oryctolagus cuniculus algirus)

Publication . Abade Dos Santos, F.A.; Dalton, Kevin P.; Carvalho, Carina L.; Casero, Maria; Alvarez, Angel L.; Parra, F.; Duarte, Margarida D.

Myxomatosis is an emergent disease in the Iberian hare (Lepus granatensis). In this species, the disease is caused by a natural recombinant virus (ha-myxoma virus [MYXV]) identified for the first time in 2018 and has since been responsible for a large number of outbreaks in Spain and Portugal. The ha-MYXV, which harbours a 2.8 Kb insert-disrupting gene M009L, can also infect and cause disease in wild and domestic rabbits, despite being less frequently identified in rabbits. During the laboratory investigations of wild leporids found dead in Portugal carried out within the scope of a Nacional Surveillance Plan (Dispatch 4757/17, MAFDR), co-infection events by classic (MYXV) and naturally recombinant (ha-MYXV) strains were detected in both one Iberian hare and one European wild rabbit (Oryctolagus cuniculus algirus). These two cases were initially detected by a multiplex qPCR detection of MYXV and ha-MYXV and subsequently confirmed by conventional PCR and sequencing of the M009L gene, which contains an ha-MYXV-specific insertion. To our knowledge, this is the first documented report of co-infection by classic MYXV and ha-MYXV strains either in Iberian hare or in European wild rabbit. It is also the first report of infection of an Iberian hare by a classic MYXV strain. These findings highlight the continuous evolution of the MYXV and the frequent host range changes that justify the nonstop monitoring of the sanitary condition of wild Leporidae populations in the Iberian Peninsula.

2022-04Journal article

Open access