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

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Saúde Animal, área científica de Sanidade AnimalABSTRACT - 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. These studies have highlighted the protective potential against hare’s myxomatosis of a commercial heterologous vaccine, particularly when the inocculated dose is increased. For monitoring and characterization of the different types of viruses responsible for myxomatosis in leporids, a multiplex qPCR system has been developed that allows for a rapid detection and differentiation between classic strains of MYXV and the natural recombinants ha-MYXV. In the course of research on myxomatosis in the Iberian hare, the presence of a herpesvirus, called Leporid gammaherpesvirus 5, was detected for the first time in this genus, for whose detection a diagnostic method based on qPCR was developed and standardized, using Evagreen systems or hydrolysis probes. The different studies carried out allowed, first of all, to realize the alarming conservation state of the wild hare in the Iberian Peninsula due to exposure to endemic and emerging pathogens, exemplified by the emergence, at least in the last three years, of naturally recombinant myxomatosis virus (ha-MYXV), also detected in wild rabbits, and the identification of a new herpesvirus in a hare, amongst other agents of bacteriological and parasitic origin, which were not the subject of this thesis. Secondly, these findings have allowed us to understand how much remains to be investigated regarding the wild animal populations in general, and the European rabbits and hares in particular. Finally, the evidence collected in this thesis supports the need for full recognition by the society in general and by the political and government entities of the value of these species for the biodiversity and the maintenance of the Mediterranean ecosystem and the very serious damages caused by their current situation..RESUMO - Título da Tese: Impacto das doenças virais dos leporídeos nos ecossistemas Ibéricos : emergência, patofisiologia, profilaxia e diagnóstico - O estado alarmante de conservação das espécies de leporídeos na Península Ibérica justifica a adopção nesta Tese de Doutoramento de uma abordagem integrativa que inclui o estudo de diferentes aspectos das interfaces vírus-hospedeiro-ambiente relativos aos principais agentes patogénicos virais do coelho-bravo (Oryctolagus cuniculus algirus) e da lebre ibérica (Lepus granatensis) nos ecossistemas mediterrânicos. Os estudos referidos nesta tese aplicaram e refinaram metodologias de biologia molecular e celular, genética, virologia, imunologia e patologia, aplicadas ao coelho-bravo e à lebre ibérica, estabelecendo-se como objectivos desta tese a investigação fisiopatológica das doenças causadas pelo vírus da doença hemorrágica do coelho (RHDV2), pelo vírus da mixomatose (MYXV) e pelo leporid gammaherpesvirus 5 (LeHV-5), os seus impactos nas espécies afetadas, o desenho de novos métodos de diagnóstico e a procura de soluções para mitigar os efeitos desses agentes nas populações selvagens de leporideos. Além de refletir sobre a correta interpretação dos diagnósticos moleculares no contexto da relação vírus-hospedeiro, como etapa preliminar à apresentação do próprio trabalho experimental, foram desenvolvidas e implementadas algumas metodologias necessárias ao seu desenvolvimento dos estudos subsequentes, como a extração de sangue através da veia jugular externa em coelho-bravo e lebre-ibérica, ou procedimentos simples para obtenção de culturas primárias de fibroblastos de leporídeos. Nesta Tese foi demonstrado que o texugo euro-asiático (Meles meles) é suscetível ao RHDV2 e que esta espécie animal pode atuar como um potencial reservatório do vírus da doença hemorrágica do coelho. A mesma estirpe de RHDV2 isolada de texugos foi identificada num coelho anão que apresentava doença prolongada (atípica) e histórico de falha vacinal com uma vacina comercial RHDV, na altura disponível no mercado português. No que diz respeito à mixomatose, esta Tese inclui a detecção e análise dos primeiros casos desta doença em lebre-ibérica, associada a uma estirpe naturalmente recombinante do vírus (ha-MYXV), bem como a primeira demonstração de que este vírus recombinante de lebres pode também infectar coelhos selvagens e domésticos. Adicionalmente, foram recolhidas evidências dos primeiros casos de co-infecção do vírus recombinante ha-MYXV e do vírus do mixoma clássico (MYXV), tanto em coelho-bravo como em lebre-ibérica. Na tentativa de fornecer soluções para o controlo da mixomatose em leporídeos selvagens, foi demonstrado que as vacinas homólogas comerciais contra a mixomatose são eficazes no coelho-bravo contra a nova estirpe naturalmente recombinante ha-MYXV mas não protegem contra a mixomatose na lebre. Esses estudos revelaram o potencial protetor de uma vacina heteróloga comercial contra a mixomatose em lebre-ibérica, principalmente quando a dose da vacina é aumentada. Para a monitorização e caracterização dos diferentes tipos de vírus responsáveis pela mixomatose em leporídeos, foi desenvolvido um sistema de qPCR multiplex que permite a rápida deteção e diferenciação entre estirpes clássicas de MYXV e ha-MYXV, recombinante natural. No decurso da investigação sobre mixomatose na lebre-ibérica, foi detectada, pela primeira vez neste género, a presença de um herpesvírus, denominado Leporid gammaherpesvirus 5, para cuja detecção foi desenvolvido e validado um método de diagnóstico baseado em qPCR, utilizando sistemas Evagreen ou sondas de hidrólise. Os diferentes estudos realizados permitiram, em primeiro lugar, reconhecer o estado alarmante de conservação da lebre-ibérica na Península Ibérica devido à exposição a múltiplos agentes patogénicos endémicos e emergentes, exemplificado pela emergência nos últimos três anos do vírus da mixomatose (ha-MYXV) naturalmente recombinante, pela detecção do mesmo vírus em coelhos selvagens e pela identificação de um novo herpesvírus em lebre, entre outros agentes de origem bacteriológica e parasitária, que não foram objeto desta tese. Em segundo lugar, essas descobertas permitiram entender o quanto ainda há a ser investigado na caracterização do estado das populações selvagens em geral e do coelho-europeu e das lebres em particular. Por último, mas não menos importante, as evidências recolhidas nesta Tese corroboram a necessidade do pleno reconhecimento por parte da sociedade civil, classe política e entidades governamentais, do valor destas espécies e das gravíssimas consequências que o seu estado atual está a ter na biodiversidade e na manutenção das espécies do ecossistema mediterrâneo.RESUMEN - Título de la tesis : Impacto de las enfermedades virales de los leporidos en los ecosistemas Ibéricos; emergencia, fisiopatología, profilaxís y diagnostico. - El estado alarmante de conservación de las especies de lepóridos en la Península Ibérica justifica la adopción en esta Tesis Doctoral de un enfoque integrador que incluya el estudio de diferentes aspectos de las interfaces virus-huésped-ambiente con respecto a los principales patógenos virales del conejo de monte (Oryctolagus cuniculus algirus) y la liebre ibérica (Lepus granatensis) en los ecosistemas mediterráneos. Los estudios a los que se refiere esta tesis han empleado y puesto a punto metodologías propias de la biología molecular y celular, la genética, la virología, la inmunología y la patología, aplicadas al conejo de monte y a la liebre ibérica, estableciéndose como objetivos de esta Tesis la investigación fisiopatológica de las enfermedades causadas por el virus de la enfermedad hemorrágica del conejo (RHDV2), el virus de la mixomatosis (MYXV) y el leporid gammaherpesvirus 5 (LeHV-5), su impacto en las especies afectadas, el diseño de nuevos métodos de diagnóstico y la búsqueda de soluciones para mitigar los efectos de estos agentes. Además de reflexionar sobre la interpretación correcta de los diagnósticos moleculares en el contexto de la relación virus-hospedador, como paso previo a la presentación de los trabajos experimentales propiamente dichos, se han puesto a punto algunas metodologías necesarias para su desarrollo, como la extracción de sangre a través de la vena yugular externa en conejos de monte y liebres ibéricas, o procedimientos sencillos para obtener cultivos primarios de fibroblastos de lepóridos. En esta Tesis se ha demostrado que el tejón euroasiático (Meles meles) es susceptible al RHDV2 y que esta especie animal puede actuar como reservorio potencial del virus de la enfermedad hemorragica del conejo, habiendose indentificado la misma cepa de RHDV2 aislada de los tejones en un conejo enano, que tenía una enfermedad prolongada (atípica) y un historial de ineficacia de vacunación con una vacuna comercial para RHDV todavía disponible en el mercado portugués. Con respecto a la mixomatosis en esta Tesis se recogen los trabajos de detección y análisis de los primeros casos de esta enfermedad en liebres ibéricas asociados a una cepa recombinante natural del virus (ha-MYXV), así como la primera demostración de que el virus recombinante de las liebres puede infectar tanto conejos de monte como domésticos. Adicionalmente se recogen las evidencias de los primeros casos de coinfección del virus recombinante ha-MYXV y el virus mixoma clásico MYXV, tanto en conejo de monte como en liebre ibérica. En un intento de aportar soluciones para el control de la mixomatosis en leporidos silvestres se ha demostrado que las vacunas homólogas comerciales contra la mixomatosis son eficaces en conejos de monte frente a la nueva cepa naturalmente recombinante ha-MYXV pero no protegen frente a la mixomatosis en la liebre ibérica. En estos estudios se ha puesto de manifiesto el potencial protector de una vacuna heteróloga comercial frente a la mixomatosis de las liebres, particularmente cuando se aumenta la dosis vacunal. Para el seguimiento y caracterización de los distintos tipos de virus responsables de la mixomatosis en leporidos se desarrolló un sistema qPCR multiplex que permite una rápida detección y diferenciación entre cepas clásicas de MYXV y los recombinantes naturales ha-MYXV. En el curso de las investigaciones sobre la mixomatosis en la liebre ibérica se detectó por primera vez en este género la presencia de un herpesvirus, denominado Leporid gammaherpesvirus 5, para cuya detección se desarrolló y puso a punto un método de diagnóstico basado en qPCR, utilizando sistemas Evagreen o sondas de hidrólisis. Los diferentes estudios realizados permitieron, en primer lugar, conocer el alarmante estado de conservación de la liebre silvestre en la Península Ibérica por exposición a patógenos endémicos y emergentes, ejemplificado por la emergencia en los últimos tres años de virus de mixomatosis (ha-MYXV) naturalmente recombinante, la detección del mismo virus en conejos de monte y la identificación de un nuevo herpesvirus en la liebre, entre otros agentes de origen bacteriológico y parasitario, que no han sido objeto de esta tesis. En segundo lugar, estos hallazgos nos han permitido comprender cuánto queda por hacer, y saber, en la investigación del estado de las poblaciones silvestres en general, y de lo conejo y liebres en particular. Por último, pero no menos importante, las evidencias que se recogen en esta Tesis apoyan la necesidad de un pleno reconocimiento por parte de la sociedad civil, la clase política y las entidades gubernamentales del valor de estas especies y las gravísimas consecuencias que su estado actual está teniendo en la biodiversidad y mantenimiento del ecosistema mediterráneo.N/

A quadruplex qPCR for detection and differentiation of classic and natural recombinant Myxoma Virus Strains of leporids

Research Areas: Biochemistry & Molecular Biology ; ChemistryA natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.info:eu-repo/semantics/publishedVersio

Nutritional value of meat lipid fraction from red-legged partridge (Alectoris rufa) obtained from wild and farmed specimens

Research Areas; Agriculture, Dairy & Animal ScienceArticle in International JournalABSTRACT - The red-legged partridge (Alectoris rufa) is a feathered game species of great socioeconomic importance in its native range and also in the UK. The aim of this study was to present a detailed comparison of meat's lipid fraction obtained from wild and farm-raised specimens and simultaneously compare the breast and leg meat portions. Meat from wild specimens had a significant (P < 0.05) lower proportion of saturated fatty acid (less 5.1%) and presented better P/S and n-6/n-3 ratios, and atherogenicity index than farm-raised counterparts. The wild specimens presented significant (P < 0.001) higher contents of total vitamin E (8.8 vs. 2.2 mu g/g of fresh meat), is for that reason less prone to lipid peroxidation than farm-raised specimens. Meat portions differed significantly (P < 0.05) on total lipid and total cholesterol contents and in all partial sums of fatty acids. The breast was leaner (0.86 vs. 1.47 g/100 g of meat), with lower total cholesterol (37.5 vs. 54.7 mg/100 g of meat), lower saturated fatty acid, monounsaturated fatty acid, polyunsaturated fatty acid, and n-6 polyunsaturated fatty acid (less 0.27, 0.28, 0.10, and 0.11 g/100 g of fresh meat, correspondingly). Regarding the fatty acid ratios and lipid quality indexes, breast meat presents better n-6/n-3 ratio and atherogenicity and thrombogenicity indexes.CIISAinfo:eu-repo/semantics/publishedVersio

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

Research Areas: Infectious Diseases ; Veterinary SciencesMyxomatosis 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.info:eu-repo/semantics/acceptedVersio

Simple method for establishing primary Leporidae skin fibroblast cultures

Research Areas: Cell BiologyCommercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal–epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.info:eu-repo/semantics/publishedVersio

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

Research Areas: Immunology ; Research & Experimental MedicineThe 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 strainsinfo:eu-repo/semantics/publishedVersio

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

Áreas de pesquisa: Infectious Diseases ; Veterinary SciencesRabbit 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 cycleinfo:eu-repo/semantics/publishedVersio

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

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.info:eu-repo/semantics/publishedVersio

A potential atypical case of rabbit haemorrhagic disease in a dwarf rabbit

ABSTRACT - Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.info:eu-repo/semantics/publishedVersio

Recombinant myxoma virus infection associated with high mortality in rabbit farming (Oryctolagus cuniculus)

ABSTRACT - Myxomatosis is an emergent disease in the Iberian hare, having been considered a rabbit disease for decades. Genome sequencing of the strains obtained from Iberian hares with myxomatosis showed these to be distinct from the classical ones that circulated in rabbits since the virus introduction in Europe, in 1952. The main genomic difference in this natural recombinant hare myxoma virus (ha-MYXV) is the presence of an additional 2.8 kb region disrupting the M009L gene and adding a set of genes homologous to the myxoma virus (MYXV) genes M060R, M061R, M064R, M065R and M066R originated in Poxviruses. After the emergence of this recombinant virus (ha-MYXV) in hares, in the summer of 2019, the ha-MYXV was not detected in rabbit surveys, suggesting an apparent species segregation with the MYXV classic strains persistently circulating in rabbits. Recently, a group of six unvaccinated European rabbits (Oryctolagus cuniculus cuniculus) from a backyard rabbitry in South Portugal developed signs of myxomatosis (anorexia, dyspnoea, oedema of eyelids, head, ears, external genitals and anus, and skin myxomas in the base of the ears). Five of them died within 24–48 hr of symptom onset. Molecular analysis revealed that only the recombinant MYXV was present. This is the first documented report of a recombinant hare myxoma virus in farm rabbits associated with high mortality, which increases the concern for the future of both the Iberian hare and wild rabbits and questions the safety of the rabbit industry. This highlights the urgent need to evaluate the efficacy of available vaccines against this new MYXV.Interdisciplinary Research Centre on Animal Health, Faculty of Veterinary Medicine, University of Lisbon (CIISA, FMV-UL), Grant/Award Number: CIISA-UIDP/CVT/00276/2020info:eu-repo/semantics/publishedVersio