Virus-host adatation and co-evolution of myxoma virus (MV) and rabbit haemorrhagic disease virus (RHDV) in their natural host, the wild rabbit (oryctolagus cuniculus)

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VIRUS-HOST ADAPTATION AND CO-EVOLUTION OF

MYXOMA VIRUS (MV) AND RABBIT HAEMORRHAGIC

DISEASE VIRUS (RHDV) IN THEIR NATURAL HOST, THE

WILD RABBIT (ORYCTOLAGUS CUNICULUS)

ALEXANDRA MÜLLER

Tese de doutoramento em Ciências Veterinárias

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3 ALEXANDRA MÜLLER

VIRUS-HOST ADAPTATION AND CO-EVOLUTION OF MYXOMA

VIRUS (MV) AND RABBIT HAEMORRHAGIC DISEASE VIRUS

(RHDV) IN THEIR NATURAL HOST, THE WILD RABBIT

(ORYCTOLAGUS CUNICULUS)

Tese de Candidatura ao grau de Doutor em Ciências Veterinárias submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto.

Orientador – Doutora Gertrude Averil Baker Thompson

Categoria – Professor Associado

Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.

Co-orientador – Doutora Paula Cristina Gomes Ferreira Proença

Co-orientador – Doutor Júlio Gil Vale Carvalheira

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Os resultados dos trabalhos experimentais incluídos na presente Tese fazem parte dos seguintes artigos científicos e publicações:

Müller, A., J. Freitas, E. Silva, G. Le Gall-Reculé, F. Zwingelstein, J. Abrantes, P. J. Esteves, P. C. Alves, W. van der Loo, Y. Kolodziejek, N. Nowotny & G. Thompson (2009). Evolution of Rabbit haemorrhagic disease virus (RHDV) in wild rabbits (Oryctolagus cuniculus) in the Iberian Peninsula. Veterinary Microbiology 135, 368-373

Müller, A. , E. Silva, J. Abrantes, P.J. Esteves, P.G. Ferreira, J.C. Carvalheira, N. Nowotny & G. Thompson (2010). Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability. Veterinary Microbiology, 140, 161-166

Müller, A. & G. Thompson (2010). Evolution of RHDV in the Iberian Peninsula: A brief review of recent findings. II Seminario Internacional sobre el Conejo Silvestre. Córdoba 28-30 Abril 2010 (in press)

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7 ACKOWLEDGEMENTS

My sincere thanks go to all colleagues that accompanied me in these years for their contribution to the present thesis, in particular to:

Professor Gertrude Thompson, for the encouragement to enrol in this postgraduate study and for her excellent supervision and guidance through the overall progress of work, for stimulating discussions, for the disposal of the Infectious Diseases Laboratory knowhow and facilities, for all the numerous opportunities given within and without this project, which contributed to the attainment of scientific maturity, and, importantly, also for all her continuous support, kind understanding and friendship at all times.

Professor Paula Ferreira and Professor Júlio Carvalheira for their co-supervision and collaboration during the development of this work, helpful discussions and for their continuous encouragement and support. Professor Artur Águas for his co-supervision and constructive suggestions, especially in the initial phases of the study.

All members of the Laboratory of Infectious Diseases, in particular: Eliane Silva and Sara Marques for sharing their expertise, technical support, aid in troubleshooting, and for their overall friendship; Jaime Freitas for his contribution to the work on RHD; Sónia Paupério, Isabel Santos, Teresa Pena, Joana Correia, Maria João Vieira, Luís Pinho, Dr. Raquel Souto for their contribution to the enriching laboratory environment. To all for the constructive lab meetings, discussions and the good moments spent together.

The Institute for Biomedical Studies (ICBAS) and the Multidisciplinary Unit for Biomedical Research (UMIB) of Porto University for infrastructural and financial support.

CIBIO for infrastructural support as well as for the permission to use valuable wild rabbit samples. Professor Pedro Esteves for the opportunity to participate in the Project on RHD and Myxomatosis (POCTI/BIA-BDE/61553/2004), him and Dr. Joana Abrantes for the interesting discussions and collaboration throughout. A special thanks to Joana for providing “hot off the press” and “hard to get” bibliography! Professor Paulo Célio Alves for interesting discussions and his persistent positive reinforcement to take up a “wild rabbit subject”.

The Zoonoses and Emerging Infections Group and all members of the Clinical Virology of the University of Veterinary Medicine, Vienna, in particular Professor Norbert Nowotny for the acceptance and supervision of the work as well as for the kind hospitality and friendship. Dr. Jolanta Kolodziejek, Helga Lussy and Hans Homola for sharing their experience and making my stay at the Clinical Virology in Vienna productive, and above all, for their friendly welcome in the group, making my stay highly enjoyable and enriching.

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Professor Támas Bakonyi, guest researcher at the Clinical Virology, Vienna, for sharing his experience on real time PCR.

Dr. Ghislaine le Gall-Recoulé, AFSSA Ploufragan, for the collaboration and constructive discussions of the joint work on RHD.

Bioportugal, Lda., in particular Dr. Joaquim Teixeira, Dr. Sónia Martins and Dr. Carla Simões for technical support in the use of the StepOne Real-time PCR.

The Laboratório de Investigação Veterinária (LNIV), Vairão, for infrastructural support, and in particular Dr. Fátima Mota for her friendship.

The Foundation for Science and Technology (FCT) for the doctoral grant (SFRH/BD/31048/2006).

And finally, my parents, for their unconditional love and support, in particular for taking care of Natália during my participation in scientific meetings and for their presence during the three month research period in Vienna.

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9 SUMÁRIO

A mixomatose e a doença hemorrágica viral (RHD) são doenças infecciosas que emergiram em populações de coelho-bravo (Oryctolagus cuniculus) na década de 1950 e 1980, respectivamente. Nos primeiros anos após o seu aparecimento, foram observadas elevadas taxas de mortalidade, mas em anos subsequentes, o impacto destas infecções parecia ter diminuído. A hipótese postulada foi de que estes vírus foram co-evoluindo com o seu hospedeiro, resultando na selecção de estirpes virais menos virulentas e de hospedeiros mais resistentes. É neste contexto, que os presentes estudos foram desenhados, visando contribuir para o conhecimento actual sobre a adaptação vírus-hospedeiro e a co-evolução do vírus da mixomatose (MV) e do vírus da doença hemorrágica viral (RHDV) ao seu hospedeiro natural, através da análise da variabilidade genética (parcial) dos vírus. Para tal, um total de 4863bp (approximadamente 3% do genoma) englobando 12 genes de nove estirpes de campo recentes de MV virulentos e de uma estirpe vacinal viva atenuada (“MAV”, Alemanha) foram sequenciadas e comparadas à estirpe virulenta originalmente introduzida “Lausanne” e ao seu derivado de campo atenuado “6918”. As nossas estirpes de campo apresentaram um máximo de três (estirpes C43, C95) e um mínimo de uma (estirpes CD01, CD05) substituições nucleotídicas em comparação com “Lausanne”. Estas estavam distribuídas ao longo de todas as regiões codificantes analisadas, excepto no gene M022L (maior proteína do envelope), onde todas as estirpes eram idênticas a “Lausanne” e “6918”. Duas novas inserções nucleotídicas simples foram observadas em algumas das estirpes de campo: na região intergénica M014L/M015L e no gene M009L, onde levou a um frameshift. Estas inserções foram localizadas após regiões homopoliméricas. A estirpe vacinal exibiu 37 substituições nucleotídicas localizadas predominantemente (95%) nos genes M022L e M036L. As regiões M009L e M014L/M015L da vacina não foram amplificadas com sucesso, sugerindo alterações genómicas maiores, que poderiam explicar o seu fenótipo atenuado. Os nossos resultados demonstraram um elevado grau de estabilidade genética de mixoma vírus (virulento) nas últimas cinco décadas. No âmbito do objectivo supracitado, também analisámos o genoma de RHDVs obtidos entre 1994 e 2007 em Portugal (40 amostras), Espanha (3 amostras) e França (4 amostras) de coelhos selvagens que sucumbiram à doença. As análises filogenéticas baseadas em sequências parciais do gene que codifica a VP60 (maior proteína estrutural do virus) permitiram um agrupamento destes RHDVs em três grupos, denominados "Grupos Ibéricos". Curiosamente, estas agruparam separadamente, embora não muito longe de RHDVs mais antigas do genogrupo 1 (contendo, por exemplo, "AST89"), mas claramente separadas de outras estirpes globais de RHDV. Este resultado deu origem à hipótese de

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que o vírus evoluiu independentemente desde a sua introdução nas populações de coelho-bravo na Península Ibérica, com os Pirenéus agindo como uma barreira natural ao movimento de coelhos e, portanto, à dispersão do vírus. Não foram observadas diferenças entre RHDV obtidas a partir de regiões geográficas onde a subespécie coelho Oryctolagus cuniculus algirus prevalece comparadas com as obtidas a partir de Oryctolagus cuniculus cuniculus. Os resultados deste trabalho foram recentemente citados por publicação internacional, na qual foi revista a origem e a filodinâmica de RHDV. A hipótese frequentemente citada sobre a coevolução vírus-hospedeiro de ambas as doenças, mixomatose e RHD, foi revista à luz dos conhecimentos actuais. Para ambas, parece ser necessário adquirir evidência adicional, que continue a apoiar esta hipótese. Finalmente, no âmbito dos trabalhos desta tese, foram desenvolvidos testes de PCR em tempo real para a detecção do RHDV e do vírus da syndrome da lebre parda (EBHSV) e apresentados os resultados preliminares do seu desempenho. Ambos os testes parecem identificar correctamente as amostras negativas, sugerindo uma alta especificidade. No entanto, algumas amostras positivas não foram correctamente identificadas, requerendo investigações adicionais e a optimização dos testes. Os trabalhos apresentados nesta tese foram desenvolvidos no âmbito do projecto “Investigação dos mecanismos que estão na base da resistência genética do coelho à mixomatose e à doença hemorrágica viral”, financiado pela Fundação para a Ciência e Tecnologia (FCT; POCTI/BIA-BDE/61553/2004) e do trabalho realizado no contexto de uma bolsa de doutoramento (SFRH/BD/31048/2006) e assim como da Unidade Multidisciplinar de Investigação Biomédica (UMIB).

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11 SUMMARY

Myxomatosis and rabbit haemorrhagic disease (RHD) are highly infectious diseases that emerged in wild European rabbit populations (Oryctolagus cuniculus) in the 1950s and 1980s, respectively. In the first years after their appearance, high mortality rates were observed, but in subsequent years, the impact of these infections seemed to have decreased. The hypothesis had been postulated that these viruses were co-evolving with their hosts, leading to the selection of less virulent strains and more resistant hosts. It is within this context, that the present studies were designed, aiming to contribute to the current knowledge on virus-host adaptation and co-evolution of myxoma virus (MV) and rabbit haemorrhagic disease virus (RHDV) in their natural host, by analysing the (partial) genetic variability of field viruses. A total of 4863bp (approximately 3% of the genome) spanning 12 genes of nine recent virulent myxoma field strains and a live attenuated vaccine strain (“MAV”, Germany) were sequenced and compared to the originally introduced virulent strain “Lausanne” and its attenuated field derivative strain “6918”. Our field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions when compared to “Lausanne”. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to “Lausanne” and “6918”. Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it lead to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of (virulent) myxoma virus over the past five decades. Within the above mentioned objective, we also analysed the genome of RHDVs obtained between 1994 and 2007 in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild rabbits that succumbed to the disease. Phylogenetic analyses based on the partial gene sequences codifying the major structural protein VP60 allowed a grouping of these RHDVs into three groups, termed “Iberian Groups”. Interestingly, these clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g. strain “AST89”), but clearly distinct from globally described RHDV strains. This result gave rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit

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subspecies Oryctolagus cuniculus algirus prevails compared with those obtained from Oryctolagus cuniculus cuniculus. The results of this work was recently cited by international publication, in which the origin and phylodynamics of RHDV analised. The frequently cited hypothesis on virus-host coevolution for both diseases, myxomatosis and RHD, was re-assessed in the light of current knowledge. For both, further evidence seems necessary further support this hypothesis. Finally, within work carried out for this thesis, Real-time PCR assays were developed for the detection of RHDV and European brown hare syndrome virus (EBHSV), and the preliminary findings on the assays performance are presented. Both assays seem to correctly identify negative samples, suggesting high specificity. However, some positive samples were not correctly identified, warranting further investigations and optimization of these assays. The studies presented in this thesis were developed within the project "Investigation of the mechanisms that underlie the genetic resistance to myxomatosis and rabbit hemorrhagic disease virus", funded by the Foundation for Science and Technology (FCT; POCTI/BIA-BDE/61553/2004), supported by a doctoral grant (SFRH/BD/31048/2006) and the Multidisciplinary Unit for Biomedical Research (UMIB).

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13 RESUMÉ

La myxomatose et la maladie hémorragique virale du lapin (RHD) sont des maladies hautement infectieuses qui ont émergé dans les populations sauvages de lapin Européen (Oryctolagus cuniculus) dans les années 1950 et 1980, respectivement. Dans les premières années après son apparition, des taux de mortalité élevés ont été observés, mais dans les années suivantes, l'incidence de ces infections semble avoir diminué. L'hypothèse a été postulée que ces virus ont co-évolué avec son hôte, résultant en la sélection de souches virales moins virulentes et d’hôtes plus résistants. C'est dans ce contexte que les études actuelles ont été conçues en vue de contribuer aux connaissances actuelles sur l'adaptation du virus-hôte et sur la co-évolution du virus de la myxomatose (MV) et du virus de la maladie hémorragique (RHDV) chez son hôte naturel, par l´analyse de la variabilité génétique (partielle) du virus. Pour ça, un total de 4863bp (environ 3% du génome) englobant 12 gènes de neuf dernières souches virulentes de MV et une souche de vaccin vivant atténué ("MAV", Allemagne) ont été séquencés et comparés à la souche virulente "Lausanne" introduite à l'origine et son dérivé atténué du champ "6918". Nos souches de terrain ont montré un maximum de trois (souches C43 et C95) et un minimum de une (souche CD01et CD05) substitutions nucléotidiques par rapport à "Lausanne". Elles ont été distribuées dans toutes les régions de codage analysées, sauf dans le gène M022L (protéine majeure d'enveloppe), où toutes les souches étaient identiques à "Lausanne" et "6918." Deux nouvelles insertions de nucléotides simples ont été observées dans certaines des souches de terrain: au sein de la région intergénique M014L/M015L et à l'intérieur du gène M009L, où elle conduit à un décalage. Ces insertions sont situées après les régions homopolymériques. La souche vaccinale affiche 37 substitutions nucléotidiques, situées principalement (95%) dans les gènes M022L et M036L. Fait intéressant, les régions M009L et M014L/M015L du vaccin n'ont pas été amplifiées avec succès, ce qui suggère des modifications majeures de la génomique qui pourraient expliquer son phénotype atténué. Nos résultats démontrent un degré élevé de stabilité génétique du virus de la myxomatose (virulent) au cours des cinq dernières décennies. Dans le objective supra-citeé, nous avons aussi analysé le génome de RHDVs obtenus, entre 1994 et 2007, au Portugal (40 échantillons), en Espagne (3 échantillons) et en France (4 échantillons), de lapins sauvages qui y avaient succombé de la maladie. Les analyses phylogénétiques basées sur des séquences partielles du gène que codifique la proteine estructurale VP60 a permis un regroupement de ces RHDVs en trois groupes, appelés " Groupes Ibériques". Fait intéressant, ces derniers ont été groupés séparément, bien que pas très loin de RHDVs du génogroupe 1 (contenant, par exemple, "AST89), mais nettement séparés des autres souches de RHDV globale. Ce

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résultat conduit à l'hypothèse que les virus ont évolué indépendamment depuis leur introduction dans les populations de lapins sauvages sur la péninsule ibérique, avec les Pyrénées agissant comme une barrière naturelle à la circulation des lapins et donc á la propagation du virus. Aucune différence n'a été observée entre le RHDV obtenu à partir de régions géographiques où la sous-espèce de lapin Oryctolagus cuniculus algirus prévaut par rapport à ceux obtenus à partir de Oryctolagus cuniculus cuniculus. Les résultats de notre travail ont été recement cités par publication internacionale sur l'origine et la phylodymamique de RHDV. L'hypothèse fréquemment citée sur la coévolution virus-hôte à la fois pour la myxomatose et RHD a été réévaluée à la lumière des connaissances actuelles. Nous avons constaté qu´il est necessaire de continuer à acquérir des éléments de preuve pour maintenir et continuer à soutenir cette hypothèse dans le cas des deux maladies infectieuses. Finalement, dans les travails de cette thèse, ont été développés tests de PCR en temps réel pour la détection de RHDV et du virus du syndrome du lièvre brun européen (EBHSV) et les résultats préliminaires de la performance des tests sont présentés. Les deux tests semblent identifier correctement les échantillons négatifs, ce qui suggère une spécificité élevée. D'autre part, certains échantillons positifs n'ont pas été correctement identifiés, justifiant de nouvelles investigations et l'optimisation de ces tests. Le travail a été élaboré dans le cadre du projet "Étude des mécanismes qui sous-tendent la résistance génétique à la myxomatose et la maladie hémorragique virale du lapin", financé par la Fondation pour la Science et la Technologie (FCT; POCTI/BIA-BDE/61553/2004) et financé par FCT grâce à une subvention de doctorat (SFRH/BD/31048/2006) et grâce au travail de l'Unité Multidisciplinaire pour la Recherche Biomédicale (UMIB).

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TABLES

Table 1 Virulence grading of myxoma virus according to Fenner and Marshall (1957) .... 36

Table 2 Selected genes and primers used for the genetic characterisation of myxoma virus field strains. The nucleotide positions refer to myxoma virus strain “Lausanne”

(GenBank accession no. AF170726) ... 54

Table 3 Observed nucleotide polymorphisms and deduced amino acid variations in recent myxoma virus field strains. The nucleotide and amino acid positions refer to myxoma virus strain “Lausanne” (GenBank accession no. AF170726) ... 56

Table 4 Genbank accession numbers of RHDV sequences included in the phylogenetic analysis ... 90

Table 5 Cycle threshold (Ct) values obtained by the application of two different primer-probe pairs in a real-time PCR assay of positive samples as determined by conventional nested RT-PCR (Moss et al., 2002) ... 112

Table 6 Comparison of diagnostic tests for the detection of European brown hare

syndrome virus (EBHSV) ... 121

Table 7 Comparison of simple and nested PCR for the detection of European brown hare syndrome virus (EBHSV) in 10-fold dilutions of samples 684/04 and 685/04 ... 122

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FIGURES

Figure 1 Monopartite, linear, single-stranded, positive-sense RNA genome of 7.3 to 8.3 kb. At 5’-terminus a virus protein (VPg)is covalently linked to genome, whereas 3’-terminus is polyadenylated (Source: ViralZone www.expasy.ch/viralzone, Swiss Institute of Bioinformatics). ... 69

Figure 2 Map of the Iberian Peninsula and South of France displaying the geographic origin of the RHDV samples analysed in this study and the time period they were collected. The distribution areas of the wild rabbit subspecies Oryctolagus cuniculus algirus and Oryctolagus cuniculus cuniculus as well as the contact zone across the Iberian Peninsula are indicated. ... 88

Figure 3 RHDV strains from Portugal cluster separately from known genogroups based on phylogenetic analysis of partial VP60 gene sequences. The neighbour joining tree was rooted with RCV. Bootstrap probability values above 75% for 1000 replicate runs are indicated at the nodes. ... 92

Figure 4 Alignment of EBHSV partial capsid gene sequences and primer-probe pairs selected for real-time PCR. The shown nucleotide positions correspond to positions 1332-1421 of the VP60 capsid gene and to positions 6563-6652 of the complete EBHSV genome (examples strain “GD”, Genbank accession numbers Z32526 and Z69629, respectively) ... 120

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“Novel infectious diseases can emerge either by a species-jump into a new host or by mutation of an existing microorganism to a more virulent form. An example of each type of emerging disease has occurred in the European rabbit (Oryctolagus cuniculus): myxomatosis, where the poxvirus myxoma virus jumped to O. cuniculus from the tapeti (a lagomorph, Sylvilagus brasiliensis), in which it caused an innocuous cutaneous fibroma, and rabbit haemorrhagic disease (RHD) where a pre-existing avirulent virus of European rabbits appears to have mutated to the lethal rabbit haemorrhagic disease virus (RHDV) that has spread around the world since 1984.” In (Kerr et al., 2009).

Myxomatosis and rabbit haemorrhagic disease (RHD) are highly infectious diseases which have emerged in wild European rabbit populations (Oryctolagus cuniculus) within the past six or seven decades. In the initial months and years after their appearance in wild and also in domestic rabbits, high mortality rates were observed. In subsequent years, the impact of these infections seemed to decrease, and the hypothesis was postulated that these viruses were co-evolving with their hosts, leading to adaptation by the selection of less virulent strains and more resistant hosts (Anderson and May, 1982; Fenner and Ross, 1994; Kerr and Best, 1998; Villafuerte et al., 1995). Much research has been carried out on this subject. In this thesis it will briefly be reviewed in the respective chapters on each disease. Some evidence has been gathered that this may be true for myxomatosis and to some extent for RHD, but knowledge on the genetic mechanisms related to host and virus is still scarce, especially for “real-life” scenario, i.e. wild rabbit populations (Best et al., 2000; Best and Kerr, 2000; Fouchet et al., 2009). This may be related, in part, to the difficulty in obtaining samples and controlling population parameters. Outbreaks in nature are typically suspected by the sudden disappearance of wild rabbits. They commonly die in their warrens. Only in areas of high rabbit density, rabbits may be found dead and eventually be sampled.

Similar to other European countries, myxomatosis and RHD have been introduced into the Iberian Peninsula in the 1950s and early 1990s, respectively (Anonymous, 1989; Monteiro, 1999; Muñoz, 1960; Villafuerte et al., 1995). Within a few years of their introduction in wild rabbit populations, both diseases caused a severe decline in rabbit abundance in the Iberian Peninsula to the extent that in Portugal the wild rabbit is currently considered a “vulnerable” species, i.e. of high risk of being extinguished (ICNB, 2005) and even as “near threatened” by the World Conservation Union in 2008 (Smith and Boyer, 2008). RHD is now considered endemic in Spain and Portugal, and despite many efforts, rabbit numbers have not fully recovered (Delibes-Mateos et al., 2008b, 2009; Dias-Pereira et al., 2004; Moreno et al., 2007; Muller et al., 2004; Santos et al., 2006; Villafuerte et al., 1995; Ward, 2005). The impacts of decreasing wild rabbit populations in Spain and Portugal are mainly twofold. On one hand, wild rabbit populations are considered a keystone species in the Iberian Mediterranean ecosystems as they represent the major food source of currently endangered specialist predators, such as the

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Iberian lynx (Lynx paradina) and the Spanish imperial eagle (Aquila adalberti) (Delibes-Mateos et al., 2008a; Moreno et al., 2004) and on the other hand, the decline of wild rabbit numbers has a severe negative economic impact on the hunting industry (Angulo and Villafuerte, 2004).

It is within this context, that a project denominated RIPAC, was developed in 2002-2004 by the Algarve´s Hunters Federation and the Regional Agricultural Directorate (RIPAC, 2004). The objectives were to determine the sanitary status and the main causes of death of small game animals, especially the wild rabbit, in the Algarve Province, Portugal. A total of 200 specimens were analysed by different elements of Porto University, including the Laboratory of Infectious Diseases, ICBAS. The presence of myxoma virus and RHDV was demonstrated in the wild rabbit subspecies Oryctolagus cuniculus algirus (Dias-Pereira et al., 2004; Muller, 2004; Muller et al., 2004; RIPAC, 2004). However the genetic characterisation of these and other virus strains from Portugal were subject of the present thesis. The overall aim of the present study is to contribute to the current knowledge on virus-host adaptation and co-evolution of MV and RHDV to their natural host, by studying the heterogeneity of selected viral genes obtained from samples taken from wild rabbits at different geographical locations in Portugal. Our studies formed part of a larger project (POCTII/BIA-BDE/61553/2004), whose goal it was to study the role of natural selection on the hypothetical increased genetic resistance of wild rabbit populations to myxomatosis and RHD.

1.1 The European wild rabbit (Oryctolagus cuniculus)

The European rabbit (Oryctolagus cuniculus) is a mammal that, together with the hare, belongs to the family Leporidae of the Order Lagomorpha. The first fossil records of lagomorphs have been attribited to the Early Paleogene, around 45 Ma (Lopez-Martinez, 2008). The first fossils of the Oryctolagus genus were dated to the Middle Pleiocene, about 3.5 Ma, from Spain and probably southern France, and those attributed to modern European rabbit species were dated to the Mid Pleistocene i.e. around 0.5 Ma (Lopez-Martinez, 2008). Based on analyses of fossil records, the Iberian Peninsula is considered the probable ancestral area of the European rabbit (Lopez-Martinez, 2008). The evolutionary history has also extensively been studied using different molecular markers such as mitochondrial DNA (Biju-Duval et al., 1991; Branco et al., 2000; Branco et al., 2002), protein polymorphism and genetic diversity on the X and Y chromossomes (Geraldes and Ferrand, 2006; Geraldes et al., 2006; Geraldes et al., 2005). Despite some

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incongruences between these different techniques, results agree that two groups of the European rabbit have been evolving in allopatry (i.e. in entirely separate ranges) during the Pleistocene (Ferrand, 2008). These correspond to the subspecies Oryctolagus cuniculus cuniculus and Oryctolagus cuniculus algirus (Ferrand, 2008). Geographically, Oryctolagus cuniculus algirus are located in the southwest and Oryctolagus cuniculus cuniculus in the northeast of the Iberian Peninsula. Both populations contact forming a line of hybridization in the central region of the Iberian Peninsula (Branco et al., 2000; Branco et al., 2002; Ferrand, 2008). From the Iberian Peninsula, and probably during the Middle Ages, O .c. cuniculus spread or was taken by humans to many other parts of continental Europe and domesticated, giving origin to different rabbit breeds (Ferrand and Branco, 2007). The geographical distribution of Oryctolagus cuniculus algirus, however, remains confined to the southwest of Spain and Portugal. Rabbits of either subspecies are not readily distinguished phenotypically, whereby molecular testing techniques currently also play an role as important conservation management tool (Esteves et al., 2006).

The European rabbit is small grey-brown mammal. It differs from the hare by smaller ears, a shorter tail and the fact that newborns are blind and furless nestlings, fully dependent on the doe. The body weight of adults ranges between 800 and 1300g (Paupério et al., 2006). The habitat consists of a mixture of pasture and scrublands as important sources of feed and shelter (Delibes-Mateos et al., 2008b). Also important is the consistence and structure of the soil, as rabbits are burrowing animals, and burrows are an essential element for social structure and reproduction (Delibes-Mateos et al., 2008b; Paupério et al., 2006). Social structure is complex and groups are generally formed by a dominant male and various female, juvenile and subordinate male animals. Social structure is strongly influenced by the habitat. Abundant feed and shelter leads to less evident hierarchy and higher reproductive indices as well as higher survival rates of juveniles. On the contrary, fragmentated or less suitable habitats lead to higher competition between individuals, more stringent social structures and in some cases to a discontinuous distribution of rabbits. Rabbits living in smaller colonies that are isolated from other colonies are also considered much more vulnerable to local extinction (Paupério et al., 2006). Each group occupies a territory of generally less than 1 hectar and rabbits normally graze within 500m of their burrows (Paupério et al., 2006). Only juveniles may disperse at longer distances. The reproductive cycle of rabbits is strongly influenced by its habitat. In mediterranean ecosystems, reproductive activities coincides with the availability of feed, i.e. Autumn, Winter and Spring (Goncalves et al., 2002; Paupério et al., 2006). The mean litter size is 4 kits per female, and each female may have 3 to 4 litters per year (Goncalves et al., 2002; Paupério et al., 2006). Juveniles leave their warren at three

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weeks of age and reach sexual maturity by 4 to 5 months. The population dynamics of wild rabbits are influenced by various factors and display seasonal fluctuations. Mortality rates are higher in juveniles that in adults. It has been estimated that up to 80% of rabbits die before reaching adulthood. Rabbit densities also vary accordingly. Higher densities are observed in Spring and early Summer, reflecting births. In Autumn, rabbit numbers decline, related to scarcity in feed, but also other factors as hunting pressure and infectious diseases such as myxomatosis and RHD (Paupério et al., 2006).

1.2 Virus-host interactions

Here, a more general approach is taken to elucidate virus-host interactions and to define related terms. More specific findings related to myxomatosis and RHD will also be reviewed in the respective disease chapters below. Viruses are small infectious agents that require living cells for replications. An infection results if a virus is able to invade and to replicate within a host. The outcome of infection frequently may vary, and thus not always results in clinical disease. Disease may result when invasion and replication of the agent and/or the host’s immune responses result in tissue damage and impair physiological functioning. The mechanisms involved vary among different host-pathogen scenarios (Mims et al., 1995). The term virulence is generally used to describe the ability of any agent to cause damage and disease and may be measured, for example, by case fatality rates or clinical scoring systems (Mims et al., 1995). Several virulence factors have been described for infectious agents, such as, e.g. Mt-7 protein for myxomatosis (Mossman et al., 1996). Host resistance is a term frequently used in the context of infectious disease, particularly in the context of RHD and myxomatosis. As for other infections, the following two situations need to be differentiated (Mims et al., 1995). On one hand, host resistance can be defined as resistance to infection, meaning that, despite exposure of a host to a particular virus, the virus is not able to infect the host. Typically, these infection-resistant animals remain seronegative despite exposure. On the other hand, it could be meaning resistance to disease, i.e. infection of the host does take place, but does not result in clinical disease. The disease-resistant host remains healthy but seroconversion occurs upon exposure. If specific antibodies are protective, this host may then be considered resistant to re-infection. The term host resistance reflects the opposite of host susceptibility. In analogy, susceptibility may mean susceptibility to either infection or disease.

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The factors determining host resistance to infection and/or clinical outcome are complex. For example, the presence of specific viral receptors on host cells is considered essential for viral attachment and entry, and thus a prerequisite for infection. In other words, resistant individuals of a susceptible species may display altered receptor configuration that do not allow virus attachment and thus infection does not occur. On the other hand, the innate immune system may play a role in preventing infection. The innate immune system includes anatomical barriers, secretory molecules as well as cellular components. As it is non-specific for a particular agent, it is also frequently termed innate resistance. In cases where successful infection occurs of a susceptible host, clinical outcome may vary considerably. In many diseases, a proportion of susceptible hosts may remain healthy (asymptomatic infection), whereas others may display mild or severe signs, and in a proportion outcome may be fatal. Different factors may have been associated to outcome of infection, such as body condition, concurrent disease, immunosupression, age, breed etc. Most of these factors do affect the immune system and as such the ability to control infection and modulate the development of disease. Both, innate and acquired immunity, are genetically programmed, as is the expression of putative viral host cell receptors. There is growing interest in genetically characterizing resistance to infection and disease. Different approaches are being taken such as the genetic characterisation of individual candidate genes up to the analysis of complete host genomes in an attempt to identify genes related with susceptibility to infectious disease (Boon et al., 2009; Brotherstone et al., 2010; Schnappinger and Ehrt, 2006; Tuite and Gros, 2006; Vidal et al., 2008).

Co-evolution of host and parasite (including viruses) does occur, when these complex interactions take place over time, resulting in selective pressures over each. Generally speaking, hosts may be under selection pressure to escape parasitism, whereas parasites may be under selection pressure to evade host defences (Anderson and May, 1982). Depending on each host-parasite scenario, the outcomes of host-parasite co-evolution may be different. Some pathogens may evolve to be harmless to their hosts. Others, such as myxoma virus, whose selection depends on transmissibility, may be expected to evolve to intermediate or even higher values of virulence alongside increasing proportions of resistant rabbits (Anderson and May, 1982; Ross and Sanders, 1977, 1984). In most cases, infectious diseases may be important drivers in the survival and adaptation of animal populations, and in the particular context of wildlife, they may also have a considerable impact on population size and host genetic diversity (Altizer et al., 2003; Daszak et al., 2000; O'Brien and Evermann, 1988; Smith et al., 2009).

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The impact and the effects of myxomatosis and RHD in European wild rabbit populations have been subject to many investigations (Boots et al., 2004; Delibes-Mateos et al., 2008b; Forrester et al., 2003; Fouchet et al., 2009; Queney et al., 2000). There is field evidence of viral attenuation as well as increasing genetic resistance in some rabbits, but underlying genetic features are far from being fully understood. Some studies aiming to identify host factors related to disease resistance have been published. These found unique changes in the chemokine receptors CXCR4 and CCR5 of Oryctolagus compared to other lagomorph members, suggesting that these may be major candidates related to resistance to myxomatosis (Abrantes et al., 2010; Abrantes et al., 2008a; Carmo et al., 2006). On the other hand, resistance to RHD has been linked to the presence of ABH blood group antigens and the presence of non-functional alleles of fucosyltransferase genes such as Fut2, determining a so-called “nonsecretor phenotype” possibly resistant to RHD (Guillon et al., 2009; Ruvoen-Clouet et al., 2000). For both diseases, these candidate host resistance factors require further studies using infectious virus under controlled conditions.

1.3 Aim and objectives

The overall aim of the present study is to contribute to the current knowledge on virus-host adaptation and co-evolution of MV and RHDV to their natural host, the wild rabbits.

Specific objectives were:

1- To genetically characterize viral strains of myxoma virus and rabbit haemorrhagic disease virus obtained from wild rabbits from different geographical locations of Portugal. 2- To assess genetic variability of selected viral genes and to compare our findings with those obtained for other European and international strains.

3- To correlate our findings with those obtained of the genetic variation of host cell receptors in order to approach the question related to viral and/or host adaptation and co-evolution.

4- To develop real-time PCR assays for the detection of RHDV and EBHSV.

This manuscript is outlined into the following sections. The next two chapters are dedicated to each disease: myxomatosis and RHD. Within each, a general literature review is presented, followed by the original research carried out. These are followed by a general discussion, in which the role of recent findings for virus-host co-evolution of both diseases will be addressed. Finally, conclusions and perspectives for future research are presented.

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Before the introduction of MV into the European wild rabbit in Europe and in Australia, interest in myxomatosis was mainly limited to some members of the scientific community (E.g. Hobbs, 1928; Hurst, 1937). But ever since the outbreaks of myxomatosis among European wild rabbits (Oryctolagus cuniculus) in Australia in 1950 and in Europe in 1952, interest exploded, not only of the larger scientific community but also of the general public. There are two major issues related with myxomatosis. First, it is the only example of the use of an infectious agent as a biological control weapon to eradicate a vertebral animal species considered a “pest”, especially in Australia where rabbits compete with autochtonous flora and fauna and also cause major agricultural losses. And second, this infection with a very lethal virus in a large population of highly susceptible mammals provided opportunities to observe the course of virus-host interaction, i.e. provided a model system to study the evolution of an infectious disease agent, and the effects of this infectious disease on the evolution of a mammal (Anderson and May, 1982; Kerr and Best, 1998).

Numerous studies were published in scientific journals (E.g. Fenner and Chapple, 1965; Fenner and Marshall, 1957; Fenner et al., 1953; Ross and Sanders, 1977, 1984, 1987), book chapters on Myxomatosis (E.g. Fenner, 1994) and even whole books on Myxomatosis were written (E.g. Fenner and Ratcliffe, 1965). In view of these excellent scientific publications and especially reviews, which are impossible to surpass, we here aim to succinctly review key aspects of the disease and to complete these with recent findings.

2.1.1 History of the introduction

Myxomatosis was recognized as a new disease in European rabbits in 1896 in Uruguay (Sanarelli, 1898). Subsequently it caused sporadic lethal infections in domestic and laboratory rabbits in Brasil and its etiological agent was shown to be a poxvirus in 1927 by Dr. H. B. Aragão at the Oswaldo Cruz Institute in Brasil. In 1918, Dr. Aragão suggested the use of myxomatosis as a means to control wild rabbit populations in Australia. The strain that was eventually introduced in Australia was termed “Standard Laboratory Strain” (SLS) or “Moses strain”, as it was recovered from a naturally infected laboratory rabbit in Rio de Janeiro (Moses, 1911 cit. by Fenner and Ross, 1994). It had been maintained by passage in laboratory rabbits for nearly 40 years before its use in field trials in the Murray Valley, Australia, in 1950, which eventually lead to the spread of the infection over this continent (Ratcliffe et al., 1952). The mortality in rabbit populations was enormous, exceeding 99% case fatality-rate. Ever since and up to now, myxomatosis is endemic in

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Australia, and epizootics occur periodically in association with local and seasonal vector activity.

In 1952, Myxomatosis was introduced in France by Dr. P. F. Armand Delille, a paediatrician who was concerned with the excessive numbers of wild rabbits at his private estate at Maillebois. He obtained a strain of myxoma virus from a friend at the Laboratoire de Bacteriologie, Lausanne, Switzerland. This introduced virus was termed “Lausanne strain”, although it originated in Campinas, Brasil, in 1949 (Bouvier 1954, cit. by Fenner and Ross, 1994). Dr Delille released two inoculated rabbits on June 14th on his land. By the end of August 1952, virtually all rabbits on his estate were dead and further outbreaks of myxomatosis were occurring in surrounding villages. By 1954 about 90% of wild rabbits had been killed, and subsequently control measures, such as immunisations, were implemented in an attempt to limit the spread of myxomatosis (Fenner and Ross, 1994). In the following years, rabbit numbers recovered, with slight geographic variations, probably also due to environmental factors, agricultural habits and hunting pressures (Arthur et al., 1988 cit. by Fenner and Ross, 1994). The disease rapidly spread to other European countries. Myxomatosis was deliberately introduced in 1953 in Kent by a resident who had brought an infected rabbit from France (History reviewed by Bartrip, 2008). By 1955 the disease had spread over most of Britain, killing an estimated 99% of rabbits (Hudson et al., 1955 and Brown et al., 1956 cit. by Fenner and Ross, 1994). Despite large local fluctuations, rabbit numbers started to increase during the 1960s, reaching 20% of the pre-myxomatosis population in 1979 (Lloyd, 1970 and Lloyd 1981 cit. by Fenner and Ross, 1994) and about one third of the pre-myxomatosis population in the 1990s (Flowerdew et al., 1992). Case-mortality rates observed in the 1970s were between 47 and 69%, and as such much lower than during the 1950s and 1960s (Ross et al., 1989). Still, myxomatosis is nowadays considered to be an important mortality factor, contributing to the control of rabbit numbers, with autumn/winter peaks of disease reducing the numbers of rabbits present at the start of the breeding season (Ross et al., 1989). The first case of myxomatosis was reported in northern Spain in 1953 (Muñoz, 1960 cit. by Alda et al., 2009), probably appearing concomitantly in Portugal. Following the initial outbreak, wild rabbit populations in the Iberian Peninsula were reduced by over 90% (Cabezas-Díaz et al., 2005). In Spain, rabbit population density appeared to increase in the 1980s, but declined again due to the introduction of rabbit haemorrhagic disease (Calvete et al., 1997; Villafuerte et al., 1995). As in other countries, myxomatosis is now endemic in Spain and Portugal (Calvete et al., 2002a; Muller et al., 2004).

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The clinical signs of myxomatosis may vary considerably due to a variety of factors, such as host species, virus-host adaptation and attenuation of viral virulence, vector-borne transmission, ambient temperature, genetic resistant rabbits and immune status (Fenner and Marshall, 1957).

In its natural hosts, the South American tapeti (Sylvilagus brasiliensis) and the North American brush rabbit (Sylvilagus bachmani) MV only induces a benign cutaneous fibroma at the site of inoculation, reflecting the long evolutionary association between the virus and its host. On the contrary, in its evolutionary new host, the European rabbit (Oryctolagus cuniculus), myxoma virus predominantly causes a highly lethal disease, termed myxomatosis. To date, actually, two forms of disease are recognized in the European rabbit: the more frequent systemic or nodular form (E.g. Silvers et al., 2006) and the less frequent amyxomatous, atypical or respiratory form (Marlier et al., 1999; Marlier et al., 2000b). Clinical signs of the nodular (classic) form include protuberant skin lesions, blepharoconjuntivitis and oedematous swellings of the head and the genital organs. The clinical signs and high mortality rates are believed to result from multiorgan dysfunction coupled with uncontrolled secondary gram-negative bacterial infections due to a progressive failure of the host’s cellular immune response. The clinical signs of the amyxomatous or atypical myxomatosis are predominantly respiratory and mortality is not a feature. Skin nodules may appear but usually are small and in reduced numbers. As this milder clinical manifestation has mostly been reported in France and Belgium (Marlier et al., 1999; Marlier et al., 2000b), a possible link between the use of the SG33 vaccine strain and the occurrence of amyxomatous myxomatosis has been postulated (Brun et al., 1981 cit. by Marlier et al., 1999). Atypical myxomatosis has been reported in the context of vaccination the Czech Republik, but genetic analyses have shown differences between the vaccine and the field strain (Psikal et al., 2003).

In between these two extreme clinical forms (classical and atypical), a whole plethora of possible clinical outcomes has been described based on field observations and on experimental inoculations (Fenner, 1994; Fenner and Marshall, 1957; Fenner and Ross, 1994; Kerr and Best, 1998). These have been linked to the process of virus-host adaptation that occurred after the introduction of MV into European wild rabbits populations. So within a few years of the release of MV in Australia and Europe, a reduction in case-fatality rates and the occurrence of attenuated MV strains has been recorded (Fenner and Chapple, 1965; Fenner and Marshall, 1957; Kerr and Best, 1998).

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For example, in Australia, a highly attenuated field strain of myxoma virus, denominated Uriarra-2-53/1 (Ur), was isolated in 1953, only 2 years after the release of SLS. It also became apparent, that there could be a selection of genetically resistant rabbits in the field, and as a result, studies were set up for monitoring the development of genetic resistance and the virulence of MV field strains (Edmonds et al., 1975; Fenner and Chapple, 1965; Fenner et al., 1953; Ross and Sanders, 1977, 1984, 1987; Sobey, 1969). The numerous experimental inoculations carried out in this context were complex, involving virus strains obtained at different geographical and temporal points and the inoculation of so-called genetically unselected laboratory rabbits, and of so-called selected rabbits, which were directly obtained from the field or bred from survivors in the field (Reviewed in Fenner, 1994; Fenner and Ross, 1994; Kerr and Best, 1998). Based on the observation of average survival time (AST) and mortality (%) of groups of inoculated laboratory rabbits, field strains were grouped into five (I to V) virulence grades (Fenner and Marshall, 1957), as shown in Table 1.

Virus (virulence) grade Average survival time (AST) Mortality (%)

I < 13 days 100

II 13-16 days 95-99

III 17-28 days 70-95

IV 29-50 days 50-70

V Not relevant < 50

Table 1 Virulence grading of myxoma virus according to Fenner and Marshall (1957)

This classification is considered the basis for detecting and monitoring the emergence of attenuated viruses in the field. Some discrepancies were found in more recent studies. For example, work in Australia has shown that viral virulence (lethality) did not always correlate with mean survival times in rabbits taken from the different localities, suggesting that the virulence of field strains may actually be higher than previously estimated (Parer, 1995; Parer et al., 1994). These results undermine previous studies that advocated an increase in the proportion of attenuated virus strains in Australia, and as such questions the concept of virus-host co-adaptation. Further interesting observations related to the virulence grading of myxoma virus strains were made recently in a pathogenicity assessment of 20 myxoma virus field strains obtained in the 1990s in Spain (Bárcena et al., 2000). The horizontal transmission of these viruses to in-contact rabbits was as also evaluated. This study found, that the average length of disease was significantly longer in the group of rabbits infected by contact in comparison to those inoculated and suggested that the stringent experimental condition could be responsible for the observed enhanced

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severity of the disease in the subcutaneously inoculated animals. In this study, a high inoculation dose of 104 plaque forming units was used instead of 5 rabbit-infectious doses of the virus (rabbit ID50), and further, very young rabbits were used: 30 days-old instead of

at least 4 month-old. The authors concluded that the results of the contact-infected rabbits would reflect more closely the real situation in the field than those of the inoculated rabbits. Although mortality rates between both groups of animals were identical (except for one strain), the AST of the contact-infected animals were higher than in the inoculated animals. As such, most of the virus strains that were classed as virulence grade I and II according to mean survival time in the inoculated animals, would actually be classified as grade III viruses in the contact-infected animals. In another recent study involving the virulence grading of two Californian MV, the observation was made for grade I and III viruses, that survival of rabbits was not altered over a dose range of 5 to 105 rabbit ID50

although the AST was reduced by around 2 days at the highest dose (Silvers et al., 2006). These recent experiments highlight the difficulty in standardizing experimental settings for evaluating myxoma virus virulence.

The selection and emergence of attenuated virus strains has also been strongly linked to the vector-borne mode of transmission of myxomatosis. Although the virus can also spread via direct contact by the respiratory route, the most important mode of transmission is by arthropod vectors. In Australia, mosquitoes such as Culex annulirostris and Anopheles annulipes are considered important, whereas in Europe, fleas such as Spilopsyllus cuniculi seem to be the principal vectors (Bull and Mules, 1944 and Lockley, 1954 cit. by Fenner and Ross, 1994). Both, mosquitoes and rabbit fleas act as mechanical vectors. The virus adheres to their mouthparts, as they probe through infected skin. Moderately attenuated viruses such as grade III or IV are more likely to be transmitted in the field as they are present in the skin for longer periods of time. On the contrary, highly virulent viruses are only present shortly before the rabbits death, and very attenuated viruses, such as grade V strains, are only infectious during a very short period as virus replication is rapidly controlled by the hosts immune response (Edmonds et al., 1975; Fenner and Marshall, 1957; Fenner et al., 1956 cit by Kerr and Best, 1998).

Interestingly, ambient temperature also has a considerable effect on the severity of disease. High temperatures favour milder clinical signs and cold climates favour severe clinical manifestation, higher case-fatality rate and higher levels of viraemia (Marshall, 1959). This may be important in that, initially, rabbits in Australia may have survived infection with moderately attenuated viruses during early stages of evolution of resistance, and may have favoured the selection of resistant rabbits in hotter climates (Kerr and Best,

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1998). The exact mechanisms underlying this phenomenon are not fully understood. Further complicating is the fact that the assessment of virulence grades by survival rates in colder indoor laboratory conditions may have led to an underestimation of host resistance in the field. As such, viruses may favour higher recovery rates, i.e. have lower mortality, in field sites with higher ambient temperatures than in colder laboratory environment (Marshall and Douglas, 1961).

Last but not least, the severity of clinical signs induced by a given myxoma virus strain is influenced by factors linked to the individual host, which in the literature has frequently been termed “resistance” or “genetic resistance”. The emergence of resistant rabbits has been studied in parallel with the monitoring of viral virulence in numerous field observations and experimental inoculations (Fenner and Chapple, 1965; Marshall and Douglas, 1961; Ross and Sanders, 1984; Williams et al., 1990). It may be exemplified by a longitudinal study performed at Lake Urana in New South Wales, Australia (Kerr and Best, 1998; Marshall and Douglas, 1961; Marshall and Fenner, 1958). Briefly, rabbit kittens taken at different field sites were taken to a central laboratory and seronegative animals were challenged at the age of 4 months or older with myxoma viruses of known virulence. Interestingly, a decrease in mortality rates and in severity of clinical signs was observed in rabbits trapped after 2 to 3 epidemics in comparison to those taken after seven epidemics. It has been postulated that the interplay between virus and host would eventually lead to the replacement of moderately virulent strains by more virulent strains as the proportion of resistant rabbits increased, and this was postulated to have occurred in the field (Anderson and May, 1982; Bárcena et al., 2000; Ross and Sanders, 1977). Attention, though, must be paid to the limitation of the diagnostic test commonly used in the 1950s and 1960s. Seronegativity was commonly assessed by the immunodiffusion test. The use of this test in longitudinal studies revealed that specific antibodies against the soluble antigen were inconsistently detected after infection, contrasting a persistent humoral response as measured by neutralisation test or ELISA (Kerr, 1997; Williams et al., 1973). In other words, the milder disease observed in challenge-inoculated “resistant” rabbits was probably, at least partly, due to the presence of antibodies not detected by the assay. The selection of resistant rabbits by mortality due to myxomatosis is expected to cause a genetic population bottleneck, leading to a reduction in genetic variation, i.e. an increase in genetic homogeneity of rabbits. A study on the genetic structure of European wild rabbits has found high degree of genetic differentiation several sites in Great Britain (Surridge et al., 1999). The authors conclude that the existence of such a myxomatosis bottleneck is possible, and that the heterozygosity observed in present populations was caused by the rapid population growth rate of rabbits. Importantly, the interpretation of this

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study is difficult due to the inexistence of data from before the introduction of myxomatosis.

2.1.3 Aetiology and virus evolution

Myxoma virus is a member of the genus Leporipoxvirus, subfamily Chordopoxvirinae, family Poxviridae (ICTVdB, 2006). Other members of the Leporipoxvirus genus include Shope fibroma virus (SFV), hare fibroma virus and squirrel fibroma virus. There are two geographic types of myxoma virus, the South American or Brazilian myxoma virus that circulates in the jungle rabbit or tapeti (Sylvilagus brasiliensis), and that is now endemic in Europe and Australasia, and the so-called Californian myxoma virus (E.g. MSW and MSD) that circulate in the brush rabbit (Sylvilagus bachmani) in the west coast of the United States of America and the Baja peninsula of Mexico. The leporipoxvirus Shope fibroma virus is genetically and antigenically closely related to myxoma virus (Cameron et al., 1999; Willer et al., 1999). Its natural host is the eastern cottontail rabbit (Sylvilagus floridanus) in North America. As this virus does not induce disseminated disease in the European rabbit, it is widely used as immunizing agent (OIE, 2009a).

Like all poxviruses, MV has a classic brick shape replicates exclusively in the cytoplasma of infected cells. Poxvirus particles consist of an envelope acquired by budding through the host cell membrane, a surface membrane, a biconcave core that contains the genome and two lateral bodies. During their life cycle, extracellular enveloped virions (EEV) and intracellular mature virions (IMV) are produced, which contain different envelopes and are infectious, but the infection is initiated by extracellular virions (ICTVdB, 2006). The genome is not segmented and contains a single molecule of linear double-stranded DNA. The central portion of approximately 120 kb of the genome encodes approximately 100 genes that are highly conserved genes among poxviruses and which encode mostly structural and housekeeping proteins. On the terminally inverted repeats (TIR) and on the near-terminal unique regions many immunomodulatory genes are located, which are presumed to have evolved closely with the natural host. As they are involved in subverting the hosts’ immune system, they may be related with inadequate host responses in the new host, the European rabbit, eventually leading to disseminated fatal disease (Barrett et al., 2001; Cameron et al., 1999; Kerr and McFadden, 2002; Stanford et al., 2007b; Stanford et al., 2007c; Zuniga, 2003).

Genetic data on poxviral evolution and thus on myxoma virus evolution is scarce, mostly due to the very large size of the viral genome. Currently the complete genome sequences of only two strains, the virulent strain ‘‘Lausanne’’, introduced in 1952 in Europe and its

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naturally attenuated field derivative ‘‘6918’’, obtained in 1995 in Spain are available (Bárcena et al., 2000; Cameron et al., 1999; Morales et al., 2009). Partial sequence information on other strains such as the Californian myxoma MSD and MSW (Jackson et al., 1999; Labudovic et al., 2004) and two Greek isolates (Kritas et al., 2008) are also available. Very recently, i.e. concomitantly with our work, partial sequence analysis of 97 field strains from 12 localities in Spain have shown an extremely low genetic variability of myxoma virus (Alda et al., 2009). Altogether, current information is still scarce to allow more accurate phylogenetic analyses and inferences about myxoma virus evolution in its new host, the European rabbit. Additionally, horizontal gene transfer (HGT) occurs in poxviruses including myxoma virus, potentially confounding phylogenetic inferences based on one or few genes (Bratke and McLysaght, 2008; Gubser et al., 2004; Hughes and Friedman, 2005; Kerr et al., 2010; Xing et al., 2006). Therefore whole-genome based phylogenetic analyses may be considered more appropriate. Whole genome comparisons between both myxoma virus strains as well as with shope fibroma virus have yielded important findings. The complete genome sequencing of Lausanne has shown that it is 161773 nucleotides long and contains a total of 171 open reading frames (ORF) encoding structural and non-structural proteins (Cameron et al., 1999). Twelve of the ORFs exist in two copies, one at each end of the TIR of 11.5kb (Cameron et al., 1999). The genome comparison of the virulent MV strain “Lausanne” and its attenuated field derivative “6918” has identified a total of 73 differences consisting of 67 base substitutions, 4 deletions and 2 insertions (Morales et al., 2009). Importantly, four disrupted genes were identified as putative determinants for the attenuation of 6918, by order of decreasing likelihood: M135R, M148R, M009R and M036L. The comparison of virulent MV Lausanne and the related apathogenic Shope fibroma virus has also shown significant differences. Eleven genes are predominantly truncated or fragmented in Shope fibroma virus suggesting that they may have possible roles in myxoma virus virulence (Cameron et al., 1999; Willer et al., 1999). Variation at other loci is also present. The role of these genes for virulence is difficult to assess (Cameron et al., 1999). Other techniques, based on the determination of restriction fragment length polymorphisms (RFLPs) have been used for the characterisation of myxoma virus field strains (Dalton et al., 2009; Kerr et al., 2010; Russell and Robbins, 1989; Saint et al., 2001). This technique is sensible enough to identify restriction patterns that could be linked to specific polymorphisms between strains. Multiple genetic types of myxoma virus were found during epidemics, which apparently were easily be replaced by others over time (Kerr et al., 2010). However, additional studies are required to evaluate the suitability of mutations determined by this technique for phylogenetic studies. No distinct correlation was found between RFLP typing and viral virulence (Kerr et al., 2010). Antigenically, most strains seem to share epitopes. High

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antigenic similarity between myxoma virus field strains have been shown by virus neutralisation tests on the chorioallantoic membrane of developing chick embryos and by immunodiffusion (Fenner and Marshall, 1957). But differences seem to exist, especially between Australian field strains and the virulent strain Lausanne, as shown by challenge experiments (Williams et al., 1973).

2.1.4 Pathogenesis and immunology

The pathogenesis of myxomatosis has been characterised in the past (Fenner and Woodroofe, 1953 cit. by Best and Kerr, 2000) and also more recently by experimental inoculations and comparative immunopathological studies (Best et al., 2000; Best and Kerr, 2000). In the latter, the virulent strain SLS and its attenuated derivative Ur were studied in genetically susceptible (laboratory) and in genetically resistant (wild) rabbits (Best et al., 2000; Best and Kerr, 2000). Briefly, rabbits were inoculated intradermally with 100PFUs in the metatarseal region of the left hind foot. Both, laboratory and wild rabbits inoculated with SLS developed clinical myxomatosis, however mortality was lower in the latter. Ur infection was characterized by moderate to severe clinical signs and occasional death in laboratory rabbits and few or no clinical signs in wild rabbits (Best and Kerr, 2000). At autopsy, several tissue samples were taken for virus titrations including the skin of the inoculation site, skin of the equivalent site of the right hind foot (distal skin), the left (draining lymph node) and right (contralateral) popliteal lymph nodes, blood, spleen and lungs. There was little difference in titres of both viruses, SLS and Ur, in the skin at the inoculation site of laboratory and wild rabbits. In the distal skin, however, virulent SLS was present in laboratory and wild rabbits by day 4 post-inoculation (p.i.), whereas as Ur was detected a few days later in laboratory rabbits and only in one of three inoculated wild rabbits. In the draining lymph nodes, either SLS or Ur viruses were present by day 2 p.i. in laboratory rabbits and by day 4 p.i. in wild rabbits. As a measure of dissemination, virus presence was also determined in the contralateral lymph node, where it was found approximately 2 days later than in the draining lymph node. Generally, SLS was slower to reach this node in wild rabbits and titres remained 10-100 times lower than in laboratory rabbits. Ur was detectable in low titres in wild rabbits only after day 15 p.i.. Neutralizing antibody responses were detectable after day 6 p.i.. Antibody titres against virulent SLS were higher and appeared earlier than those against Ur in both, laboratory and wild rabbits. This work showed, that neither attenuation nor resistance were initially strongly manifested in the skin at the inoculation site, but were more evident at the distal skin site, and that the draining lymph node was a critical organ for amplification and dissemination of the virus and thus for disease outcome.