We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes.

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The protozoan Plasmodium vivax is responsible for 42% of all cases of malaria outside Africa. The parasite is currently largely restricted to tropical and subtropical latitudes in Asia, Oceania, and the Americas. Though, it was historically present in most of Europe before being finally eradicated during the second half of the 20th century. The lack of genomic information on the extinct European lineage has prevented a clear understanding of historical population structuring and past migrations of P. vivax. We used medical microscope slides prepared in 1944 from malaria-affected patients from the Ebro Delta in Spain, one of the last footholds of malaria in Europe, to generate a genome of a European P. vivax strain. Population genetics and phylogenetic analyses placed this strain basal to a cluster including samples from the Americas. This genome allowed us to calibrate a genomic mutation rate for P. vivax, and to estimate the mean age of the last common ancestor between European and American strains to the 15th century. This date points to an introduction of the parasite during the European colonization of the Americas. In addition, we found that some known variants for resistance to antimalarial drugs, including Chloroquine and Sulfadoxine, were already present in this European strain, predating their use. Our results shed light on the evolution of an important human pathogen and illustrate the value of antique medical collections as a resource for retrieving genomic information on pathogens from the past.

BackgroundInternational travel and immigration have been related with an increase of imported malaria cases. This fact and climate change, prolonging the period favouring vector development, require an analysis of the malaria transmission resurgence risk in areas of southern Europe. Such a study is made for the first time in Spain. The Ebro Delta historically endemic area was selected due to its rice field landscape, the presence of only one vector, Anopheles atroparvus, with densities similar to those it presented when malaria was present, in a situation which pronouncedly differs from already assessed potential resurgence areas in other Mediterranean countries, such as France and Italy, where many different Anopheles species coexist and a different vector species dominates.MethodsThe transmission risk was assessed analysing: 1) climate diagrams including the minimum temperature for Plasmodium falciparum and Plasmodium vivax development; 2) monthly evolution of the Gradient Model Risk (GMR) index, specifying transmission risk period and number of potential Plasmodium generations; 3) ecological characteristics using remote sensing images with the Eurasia Land Cover characteristics database and the monthly evolution of the Normalized Difference Vegetation Index (NDVI); 4) evaluation of A. atroparvus population dynamics.ResultsClimatological analyses and GMR index show that a transmission risk presently exists, lasting from May until September for P. falciparum, and from May until October for P. vivax. The GMR index shows that the temperature increase does not actually mean a transmission risk increase if accompanied by a precipitation decrease reducing the number of parasite generations and transmission period. Nevertheless, this limitation is offset by the artificial flooding of the rice fields. Maximum NDVI values and A. atroparvus maximum abundance correspond to months with maximum growth of the rice fields.ConclusionsThe Ebro Delta presents the ecological characteristics that favour transmission. The temperature increase has favoured a widening of the monthly potential transmission window with respect to when malaria was endemic. The combined application of modified climate diagrams and GMR index, together with spatial characterization conforms a useful tool for assessing potential areas at risk of malaria resurgence. NDVI is a good marker when dealing with a rice field area.

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe—where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum. Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.