Quadro anatomo-histopatológico e diagnóstico molecular da doença hemorrágica viral em coelho-bravo

Dissertação de Mestrado Integrado em Medicina VeterináriaO vírus da doença hemorrágica dos coelhos de tipo 2 (RHDV2) foi detetado em Portugal pela primeira vez, em 2012, e encontra-se atualmente disseminado em todo o território nacional incluindo Madeira, Açores e Berlengas. O papel ecológico e económico do coelho-bravo, aliado à sua importância para os níveis tróficos superiores, levou a que o Governo Português ativasse em 2017 um plano para controlo desta doença (Despacho 4757/2017 de 31 de Maio). Este trabalho teve como objetivo estabelecer padrões de lesão histopatológica nos principais órgãos afetados (fígado, pulmão, baço, duodeno, coração, entre outros) durante a infeção por RHDV2 e relacioná-los com os padrões de distribuição de cargas virais (medidos através dos valores de Cq obtidos por RT-qPCR) em sete matrizes (fígado, baço, duodeno, fezes, rim, pulmão e ventrículo esquerdo). Todos os coelhos-bravos investigados (n=49), foram obtidos no âmbito deste estudo, durante suspeitas de surtos de DHV, sendo oriundos de vários locais. No grupo dos animais não vacinados, o diagnóstico virológico e histopatológico raramente suscitou dúvidas e não foram encontradas lesões macro e microscópicas diferentes entre os coelhos jovens e os animais adultos. No entanto foi observada uma diminuição significativa das cargas virais nos órgãos dos animais vacinados para RHDV2 quando comparados com os animais não vacinados, tal como já descrito na literatura. Em alguns animais vacinados, foi difícil detetar o vírus por métodos moleculares apesar da presença de graves lesões histopatológicas compatíveis com DHV. No caso dos animais vacinados, a valores de Cq tendencialmente maiores, correspondeu uma menor prevalência do padrão lesional hepático mais grave. Contrariamente, nos animais não vacinados, foram encontrados valores de Cq tendencialmente mais baixos no fígado (maiores cargas virais), correspondendo a um padrão lesional mais grave. Os dados obtidos indicam também que o fígado não é o órgão de eleição para diagnóstico de RHDV2 em animais vacinados, já que o pulmão foi a matriz onde o vírus foi mais detetado. Curiosamente, o ventrículo esquerdo apresentou-se como a matriz com maior percentagem de positividade em todos os grupos pelo que a pertinência da sua utilização sistemática no diagnóstico molecular, deve ser investigada. Foi ainda realizada a pesquisa de RHDV2 em outras espécies simpátricas: lebre ibérica (n=2), toirão (n=1), texugo (n=1), sacarrabos (n=1), roedores (n=18), pardalcomum (n=1), insetos (n> 2568) e ixodídeos (n=28)). Foi detetado RNA viral no pulmão de um toirão, no fígado e ventrículo esquerdo de uma lebre, nas fezes do pardal comum, e em 3 famílias de insetos (Ceratopogonidae, Staphylinidae e Simuliidae). Este trabalho trouxe novos dados para a compreensão da interface patogenia-diagnóstico e para a compreensão da eco-epidemiologia da DHVABSTRACT - Anatomo-histopathological analysis and molecular diagnosis of Viral Haemorrhagic Disease in wild rabbit - Rabbit haemorrhagic disease virus 2 (RHDV2) was detected in Portugal for the first time in 2012. It is currently widespread in the continent and islands (Azores, Madeira and Berlengas). The ecological and economic role of the wild rabbit and its crucial importance for the higher trophic levels, led the Portuguese Government to activate, in 2017, an action plan to control this disease (Dispatch 4757/2017 of 31 May). This work aimed to establish patterns of histopathological lesions in the organs affected during infection (liver, lung, spleen, thymus, duodenum, heart among others) and to relate them to the viral load distribution patterns, measured by the Cq values obtained with a RHDV2-specific RT-qPCR, in seven biological matrices (liver, spleen, duodenum, faeces, kidney, lung and left ventricle). All the wild rabbits investigated (n = 49) were obtained within the scope of this study and originated from several locations (mainly two wild-rabbit farms with management conditions and different purposes) during suspected outbreaks of haemorrhagic viral disease. In the group of unvaccinated animals, the virological and histopathological diagnosis rarely raised doubts and no different macro and microscopic lesions were found between young rabbits and adult animals No differences in the macro and microscopic lesions were found between young and adult rabbits but a significant decrease in the viral loads of organs was observed in RHDV2 vaccinated rabbits, when compared to non-vaccinated rabbits, as described before in the literature. In some animals that had been vaccinated, it was difficult to detect the virus by molecular methods, although severe histopathological lesions were identified. For the vaccinated group, the increase in the Cq values tended to be accompanied by a more variable lesional pattern, with the more severe ones being less prevalent. In the liver of nonvaccinated rabbits, for example, as Cq values decreased (corresponding to higher viral charges), more severe lesional patterns were observed. Our data indicate that the liver is not the organ of choice for RHDV2 molecular diagnosis in vaccinated animals, since the virus was more often detected in the lungs. Interestingly, the left ventricle showed the highest percentage of positivity in all groups, so the relevance of its systematic use for diagnosis should be considered and investigated. RHDV2 was also investigated in other sympatric species, such as Iberian hare (n=2), toad (n=1), badger (n=1), rodents (n=18), common sparrow (n=1), insects (n>2568) and ixodids (n=38). Viral RNA was detected in the lung of a toad, in the liver and heart of a hare in the faeces of the common sparrow, and in 3 families of insects (Ceratopogonidae, Staphylinidae and Simuliidae). This work brought new insights to the pathogeny-diagnosis interface and to the comprehension of the disease eco-epidemiology.Financiado por: Fundo Florestal PermanenteN/

Harmless or Threatening? Interpreting the Results of Molecular Diagnosis in the Context of Virus-Host Relationships

Molecular methods, established in the 1980s, expanded and delivered tools for the detection of vestigial quantities of nucleic acids in biological samples. Nucleotide sequencing of these molecules reveals the identity of the organism it belongs to. However, the implications of such detection are often misinterpreted as pathogenic, even in the absence of corroborating clinical evidence. This is particularly significant in the field of virology where the concepts of commensalism, and other benign or neutral relationships, are still very new. In this manuscript, we review some fundamental microbiological concepts including commensalism, mutualism, pathogenicity, and infection, giving special emphasis to their application in virology, in order to clarify the difference between detection and infection. We also propose a system for the correct attribution of terminology in this context.info:eu-repo/semantics/publishedVersio

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021

Funding Information: This work received financial support from the Global Health and Tropical Medicine Center (which is funded through Fundação para a Ciência e a Tecnologia (FCT) contract UID/Multi/04413/2013). This research was also funded by FCT, Project UIDB/00276/2020 and LA/P/0059/2020-AL4AnimalS, and by the Interdisciplinary Research Center on Animal Health (CIISA), Faculty of Veterinary Medicine, University of Lisbon (Portugal). Finally, this research was also partially funded by the Interdisciplinary Research Center on Animal Health (Project CIISA-INOV 4/2021), Faculty of Veterinary Medicine, University of Lisbon (CIISA, FMV-UL) (Portugal). Publisher Copyright: © 2023 by the authors.In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.publishersversionpublishe

Leporids’ Emerging Diseases as a Threat to Biodiversity

Wild leporids have been gaining interest and prominence in the scientific and social community worldwide. While endangered of extinction in its native territory, the Iberian Peninsula, where it has a key role in the Mediterranean ecosystems, the European rabbit (Oryctolagus cuniculus) is considered a plague in Australia, due to the great economic and ecological consequences of its presence in the territories. The impact of viral diseases on the Leporidae family’ members, namely on the European rabbit, has been largely recognized worldwide since the early 50s, due to the emergence of myxomatosis and, from the mid-80s onwards, due to the emergence of rabbit haemorrhagic disease virus 1 and 2. More recently, in 2018, a recombinant myxoma virus emerged with the ability to infect and cause severe disease in the Iberian hare (Lepus ganatensis). Also, a new gammaherpesvirus was described in Iberian hares, associated with myxoma virus infections. In this chapter, we revise the main viral infectious treats to the native leporids of the Iberian Peninsula. The recovery of the European rabbit populations, as well as of other leporid species around the world, is currently a major challenge for the scientific and social communities and policy-makers. If we fail, the ripple effects on the trophic web will be so dramatic that are likely to be unrecoverable

The Health and Future of the Six Hare Species in Europe: A Closer Look at the Iberian Hare

Although there are around 40 species of hares in the world divided into three different genera (Lepus, Caprolagus, and Pronolagus), only six species inhabit Europe, all belonging to genus Lepus. The conservation status of these six species was recently revised in the International Conservation Union (IUCN) Red List of Threatened Species. Lepus castroviejoi and L. corsicanus were attributed the status of “vulnerable”. The other four species, L. europaeus, L. timidus, L. capensis, and L. granatensis, were considered of “least concern” although a declining trend was recognized for the last two species’ wild populations. Here we review the major threats to the hare species in Europe, with emphasis on infectious diseases. Furthermore, we present the sanitary data regarding the Iberian hare populations from Portugal, which were severely affected by the emergence of a naturally occurring recombinant myxoma virus (MYXV), first reported in mid-2018. The recent detection in 2019 of a leporid herpesvirus (LeHV-5), which pathogenicity appears to be exacerbated in MYXV-infected hares, brings additional concerns to the health and conservation of the Iberian hare

Bagaza virus in wild birds, Portugal, 2021

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.Peer reviewe

Iberian hare and European rabbit extinctions: the foundation of a Breeding Centre in Portugal

Introduction: The Iberian hare (Lepus granatensis) and wild rabbit (Oryctolagus cuniculus algirus) are endemic and key species, that have declined the hare by more than 50% in the last four years after the emergence of a natural recombinant myxoma virus (ha-MYXV) and the putative reactivation of Leporid gammaherpesvirus 5 (LeHV-5) which together will lead to the reclassification of its conservation status to Vulnerable by the IUCN this year. Material & Methods: Physical and technical facilities (about 2 ha) were created including breeding, feeding and physical training parks as well as methods for disinfection, deworming, and capture of animals with minimal stress, and the specific probe was built for the Iberian hare for semen collection. Results: Currently, the centre has 4 epidemiological units with 60 hares and 2 epidemiological units with 40 wild rabbits (Endangered, IUCN). During this year, repopulations were started (43 hares and 67 rabbits) in Évora with 2.7% of mortality.The eletrojeaculaton method has allowed the collection of sperm in about 5-10 minutes, of a concentration of 1.06x109±7.98x108 spz/mL. Discussion:  In the next years the annual restocking will include 200 hares and 300 rabbits. Artificial insemination will be started and it is expected that improve the species' prolificacy, increasing the number of animals released into the wild.   Keywords: Iberian hare; European rabbit; Conservation medicine; Reproduction.   Funding: The work is conducted at the Lepus Organization (www.lebre-iberica.pt), supported by the company FAASNature, Lda - Conserving wildlife for a thriving tomorrow

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids

A 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

Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994–1999 and 2017–2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994–1999, none MYXV-DNA positive, and n = 28 sampled in 2017–2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018–2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017–2019 (13.0%, CI95% 9.2–18.2%) than in 1994–1999 (5.4%, CI95% 3.0–9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018–2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.This work was financed by the Fundo Florestal Permanente, Government of Portugal, (Project +Coelho 2, ref. 2019014300001, as part of the Action Plan for the Control of Rabbit Viral Haemorrhagic Disease (Dispatch no. 4757/2017 of 31 May) and by the Spanish Ministry of Science and Innovation (PID2019-111080RB-C21). Fundação para a Ciência e Tecnologia (FCT) funded Nuno Santos (grant SFRH/BPD/116596/2016) and Fábio A. Abade dos Santos (SFRH/BD/137067/2018). The Centre for Interdisciplinary Research in Animal Health (CIISA) from the Faculty of Veterinary Medicine (University of Lisbon) also contributed to this study (CIISA-UIDP/CVT/00276/2020, LA/P/0059/2020 – AL4AnimalS). MMH was funded by Junta de Comunidades de Castilla-La Mancha and the European Regional Development Fund (SBPLY/17/180501/000514).Peer reviewe

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species