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

Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples

Staphylococcus epidermidis is considered to be one of the most common causes of nosocomial bloodstream infections, particularly in immune-compromised individuals. Here, we report the development and application of a novel peptide nucleic acid probe for the specific detection of S. epidermidis by fluorescence in situ hybridization. The theoretical estimates of probe matching specificity and sensitivity were 89 and 87%, respectively. More importantly, the probe was shown not to hybridize with closely related species such as Staphylococcus aureus. The method was subsequently successfully adapted for the detection of S. epidermidis in mixed-species blood cultures both by microscopy and flow cytometry.This work was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (PhD Fellowship SFRH/BD/29297/2006 and Post-Doc Fellowship SFRH/BPD/42208/2007)

A survey of the eutrophication state of an urbanized tropical estuary, the case of the Great Vitória Estuarine System, Brazil.

Although, estuarine ecosystems have an ecological and economical importance as they have a high  biological productivity and occur within a unique and dynamic environment, they have been subjected to anthropogenic alterations. The Great Vitória Estuarine System (GVES), Espírito Santo State, Brazil, is not an exception, as urbanization is growing around it with a high quantity of sewage been added to the system. In order to evaluate the eutrophication state as well as the meiofauna response to it, several parameters in both sediments and in the water column were assessed. Orthophosphate, nitrite, nitrate, ammonia, chla and Fecal Coliform counts (FC) ranged from 0.2 to 3.2 μM, 0.25 to 1.14 μM, 1.83 to 0.19μM, 4.19 to 49.23 μM, 0.61 to 6.72 μg/L and 14 to 5.0x104 MPN/100 mL of water, respectively. These results showed that the GVES is under an eutrophication process and that the Passagem Channel experienced the largest impacts. Sewage plays an important role in this eutrophication process as indicated by PCA and correlations tests. The density of meiofauna showed similar values to those found in environments with similar levels of anthropogenic stress. This multi-approach evaluation revealed several aspects of the impacted estuary and could be used as an important tool to manage better the estuary.Although, estuarine ecosystems have an ecological and economical importance as they have a high  biological productivity and occur within a unique and dynamic environment, they have been subjected to anthropogenic alterations. The Great Vitória Estuarine System (GVES), Espírito Santo State, Brazil, is not an exception, as urbanization is growing around it with a high quantity of sewage been added to the system. In order to evaluate the eutrophication state as well as the meiofauna response to it, several parameters in both sediments and in the water column were assessed. Orthophosphate, nitrite, nitrate, ammonia, chla and Fecal Coliform counts (FC) ranged from 0.2 to 3.2 μM, 0.25 to 1.14 μM, 1.83 to 0.19μM, 4.19 to 49.23 μM, 0.61 to 6.72 μg/L and 14 to 5.0x104 MPN/100 mL of water, respectively. These results showed that the GVES is under an eutrophication process and that the Passagem Channel experienced the largest impacts. Sewage plays an important role in this eutrophication process as indicated by PCA and correlations tests. The density of meiofauna showed similar values to those found in environments with similar levels of anthropogenic stress. This multi-approach evaluation revealed several aspects of the impacted estuary and could be used as an important tool to manage better the estuary

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

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

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio