Novel Nonnucleoside Inhibitors of Zika Virus Polymerase Identified through the Screening of an Open Library of Antikinetoplastid Compounds.

Zika virus (ZIKV) is a mosquito-borne pathogen responsible for neurological disorders (Guillain-Barré syndrome) and congenital malformations (microcephaly). Its ability to cause explosive epidemics, such as that of 2015 to 2016, urges the identification of effective antiviral drugs. Viral polymerase inhibitors constitute one of the most successful fields in antiviral research. Accordingly, the RNA-dependent RNA polymerase activity of flavivirus nonstructural protein 5 (NS5) provides a unique target for the development of direct antivirals with high specificity and low toxicity. Here, we describe the discovery and characterization of two novel nonnucleoside inhibitors of ZIKV polymerase. These inhibitors, TCMDC-143406 (compound 6) and TCMDC-143215 (compound 15) were identified through the screening of an open-resource library of antikinetoplastid compounds using a fluorescence-based polymerization assay based on ZIKV NS5. The two compounds inhibited ZIKV NS5 polymerase activity in vitro and ZIKV multiplication in cell culture (half-maximal effective concentrations [EC50] values of 0.5 and 2.6 µM for compounds 6 and 15, respectively). Both compounds also inhibited the replication of other pathogenic flaviviruses, namely, West Nile virus (WNV; EC50 values of 4.3 and 4.6 µM for compounds 6 and 15, respectively) and dengue virus 2 (DENV-2; EC50 values of 3.4 and 9.6 µM for compounds 6 and 15, respectively). Enzymatic assays confirmed that the polymerase inhibition was produced by a noncompetitive mechanism. Combinatorial assays revealed an antagonistic effect between both compounds, suggesting that they would bind to the same region of ZIKV polymerase. The nonnucleoside inhibitors of ZIKV polymerase here described could constitute promising lead compounds for the development of anti-ZIKV therapies and, eventually, broad-spectrum antiflavivirus drugs.

Pneumocystis jirovecii and microsporidia: An unusual coinfection in HIV patients?

Pneumocystis jirovecii and microsporidia species are recognized as opportunistic infectious pathogens in AIDS patients. Coinfection of both in one patient has been rarely reported. The aim of the present study was to investigate the coinfection of P. jirovecii and microsporidia in different tissues from AIDS deceased patients. Post mortem histological finding of P. jirovecii and microsporidia was demonstrated by means of the Grocott's methenamine silver and Brown Brenn staining, respectively. Molecular technique was used for identification and characterization of both fungi. Out of the 514 autopsied cases P. jirovecii and microsporidia species were identified in 53 (10.3%) and 62 (12.1%) cases respectively. A total of five cases (0.97%) coinfected with Pneumocystis and microsporidia were recovered from all analyzed autopsies. Coinfection of Pneumocystis and microsporidia is very challenging and raises interesting issues about host-parasite relationship. The early diagnosis of both pathogens must be crucial to establish correct and early treatments, improve the patient's evolution, reducing the risk of death.

Genotypes of Cryptosporidium spp. and Enterocytozoon bieneusi in Human Immunodeficiency Virus-Infected Patients in Lagos, Nigeria.

Molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi has improved our understanding of the transmission of both organisms in humans. In this study, to infer possible infection sources, Cryptosporidium spp. and E. bieneusi in fecal specimens from 90 HIV-infected patients attending antiretroviral clinics in Lagos, Nigeria were detected and genotyped by PCR and DNA sequencing. Cryptosporidium spp. and E. bieneusi were identified in four and five patients, respectively, including the occurrence of subtype IeA11T3G3 of Cryptosporidium hominis in two patients, subtype IIcA5G3k of Cryptosporidium parvum in one patient, and Type IV of E. bieneusi in four patients. Among the remaining positive patients, one had mixed infection of Cryptosporidium meleagridis and C. hominis and one had mixed E. bieneusi genotypes. These data highlight a possible difference in major transmission routes (anthroponotic vs. zoonotic) between Cryptosporidium spp. and E. bieneusi in HIV+ patients in the study area.

Capillary electrophoresis reveals polyamine metabolism modulation in Leishmania (Leishmania) amazonensis wild-type and arginase-knockout mutants under arginine starvation.

l-Arginine is an essential amino acid in Leishmania (Leishmania) amazonensis metabolism. A key enzyme for parasite l-arginine metabolism is arginase (ARG) that uses arginine to produce urea and ornithine, a precursor of polyamine pathway guaranteeing parasite replication in both insect and mammal hosts. There is an alternative pathway to produce ornithine via l-proline and glutamate, but this mechanism is not described in Leishmania. In the mammal host, two enzymes can use l-arginine as substrate, the host ARG and the induced nitric oxide synthase that produces nitric oxide. The competition between induced nitric oxide synthase and both parasite and host ARG can favor the success of the infection or its control. Here, we established the metabolomics profile of the polyamine pathway of wild type (WT) L. (L.) amazonensis, submitted or not to l-arginine starvation, and compared to the ARG-knockout mutant (arg- ). Our results indicated that arginine starvation induces a decrease in arginine, ornithine, and putrescine, but we could not detect the significative level changes of spermidine, spermine, or agmatine. However, the absence of ARG on the arg- induced an increase of arginine and citrulline levels, but decreased the levels of ornithine and putrescine. Similarly to the WT arginine-starved parasites, the arg- parasites presented lower levels of proline when compared to the WT ones. This could be indicative of an alternative pathway to surpass the enzyme or its substrate absence.

Detection of biomarkers of pathogenic Naegleria fowleri through mass spectrometry and proteomics.

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.

Molecular characterization of human-pathogenic microsporidia and Cyclospora cayetanensis isolated from various water sources in Spain: a year-long longitudinal study.

Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

[Molecular characterization of Cryptosporidium spp. isolated in humans in two different locations in Spain]. / Caracterización molecular de aislados humanos de Cryptosporidium spp. procedentes de 2 diferentes localizaciones de España.

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions.

Microsporidia in Commercially Harvested Marine Fish: A Potential Health Risk for Consumers.

Microsporidia are widely spread obligate intracellular fungal pathogens from vertebrate and invertebrate organisms, mainly transmitted by contaminated food and water. This study aims to detect the presence of major human-pathogenic microsporidia, i.e., Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi, in the gastrointestinal tract of commercially harvested marine fish from Mediterranean coast of the Comunidad Valenciana, Eastern Spain. A total of 251 fish, 138 farmed fish and 113 wild fish from commercial fishing were tested by SYBR Green real-time PCR, enabling the simultaneous detection of the four targeted species. E. intestinalis/hellem was found in 1.45% of farmed fish and 7.96% of wild fish, while Enterocytozoonidae was detected in 2.90% and 18.58% of farmed and wild fish, respectively. E. cuniculi was not detected in any of the analyzed specimens. To the authors' knowledge, this is the first report of E. intestinalis/hellem in fish, particularly in marine fish. Although the role of fish in these species' epidemiology remains unknown, this finding points out a potential public health risk linked to fish consumption. Further studies are necessary to characterize these microsporidia in fish hosts better and to elucidate their epidemiological role.

Identification of West Nile virus RNA-dependent RNA polymerase non-nucleoside inhibitors by real-time high throughput fluorescence screening.

West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 µM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1-4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 µM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors.

Microsporidia as a Potential Threat to the Iberian Lynx (Lynx pardinus).

Lynx pardinus is one of the world's most endangered felines inhabiting the Iberian Peninsula. The present study was performed to identify the presence of microsporidia due to the mortality increase in lynxes. Samples of urine (n = 124), feces (n = 52), and tissues [spleen (n = 13), brain (n = 9), liver (n = 11), and kidney (n = 10)] from 140 lynxes were studied. The determination of microsporidia was evaluated using Weber's chromotrope stain and Real Time-PCR. Of the lynxes analyzed, stains showed 10.48% and 50% positivity in urine and feces samples, respectively. PCR confirmed that 7.69% and 65.38% belonged to microsporidia species. The imprints of the tissues showed positive results in the spleen (38.46%), brain (22.22%), and liver (27.27%), but negative results in the kidneys. PCR confirmed positive microsporidia results in 61.53%, 55.55%, 45.45%, and 50%, respectively. Seroprevalence against Encephalitozoon cuniculi was also studied in 138 serum samples with a positivity of 55.8%. For the first time, the results presented different species of microsporidia in the urine, feces, and tissue samples of Lynx pardinus. The high titers of anti-E. cuniculi antibodies in lynx sera confirmed the presence of microsporidia in the lynx environment. New studies are needed to establish the impact of microsporidia infection on the survival of the Iberian lynx.

Latent Microsporidia Infection Prevalence as a Risk Factor in Colon Cancer Patients.

Microsporidia are opportunistic intracellular parasites, generating serious pathology in individuals with a compromised immune system. Infection by microsporidia inhibits p53 and Caspase 3, proteins involved in apoptosis and the cell cycle, which are vital in the malignant process of epithelial cells. The presence of microsporidia in the intestinal tissues of 87 colon cancer (CC) patients and 25 healthy controls was analyzed by real-time PCR and an immunofluorescence antibody test. Anti-Encephalitozoon antibodies were analyzed in serum samples by ELISA (enzyme linked immunosorbent assay). In 36 (41.3%) CC cases, microsporidia infections were identified in their tissues vs. no cases among control subjects (p < 0.0001). An increase in IgG and IgE anti-Encephalitozoon antibodies was found in patients with CC, which would demonstrate continuous and previous contact with the parasite. The high prevalence of microsporidia in tissues and the seroprevalence in patients with CC suggest a relationship between microsporidia and the etiopathogenesis of CC.

Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees.

Nosema ceranae is currently one of the major pathogens of honeybees, related to the worldwide colony losses phenomenon. The genotyping of strains based on ribosomal DNA (rDNA) can be misleading if the repeated units are not identical. The analysis of cloned rDNA fragments containing the intergenic spacer (IGS) and part of the rDNA small-subunit (SSU) gene, from N. ceranae isolates from different European and Central Asia populations, revealed a high diversity of sequences. The variability involved single-nucleotide polymorphisms and insertion/deletions, resulting in 79 different haplotypes. Two sequences from the same isolate could be as different as any pair of sequences from different samples; in contrast, identical haplotypes were also found in very different geographical origins. Consequently, haplotypes cannot be organized in a consistent phylogenetic tree, clearly indicating that rDNA is not a reliable marker for the differentiation of N. ceranae strains. The results indicate that recombination between different sequences may produce new variants, which is quite surprising in microsporidia, usually considered to have an asexual mode of reproduction. The diversity of sequences and their geographical distribution indicate that haplotypes of different lineages may occasionally be present in a same cell and undergo homologue recombination, therefore suggesting a sexual haplo-diploid cycle.

[Cryptosporidium: a genus in revision. The situation in Spain]. / Cryptosporidium: un género en revisión. Situación en España.

Genus Cryptosporidium, has undergone major revisions in recent years. The identification of new species and their major reservoirs has contributed to the knowledge of the epidemiology of human infection. In Spain, although there are many publications, few studies have been conducted to identify the circulating species and genotypes. This fact has led us to review and update these new studies published in Spain, particularly those that use molecular methods in order to characterise the species and genotypes present in our country.

Are pathogenic Legionella non-pneumophila a common bacteria in Water Distribution Networks?

The present study analyzes at the national level, the presence of circulating Legionella in the artificial aquatic systems of different facilities of all of them state-owned centers throughout Spain for 12 months. 1754 water samples from various state-owned centers were collected from January to December 2014. Samples were collected from the cooling towers and evaporative condensers (CTC), and water distribution networks such as domestic hot water (DHW), cold water for human consumption (CW), sprinkler irrigation systems (SIS), fire sprinkler systems (FSS), and water from decorative fountains (DF). All these facilities are considered, according to current regulations, as potential amplifying systems for bacteria and possible sources of infection by the generation of droplets and aerosols. The isolation and counting of Legionella in water samples was carried out using microbiological culture following the international normative UNE-EN-ISO 11,731:2007 (ISO 11,731:1998) and UNE-EN ISO 8199:2008 (ISO 8199:2005).The quantification of Legionella colonization, the annual distribution, and the geographical distribution of the Legionella isolates recovered in the water were analyzed. Besides, molecular techniques were used for the characterization of the Legionella non-pneumophila isolates. Legionella was recovered from 15.79% of the analyzed water samples. High colonization was more frequently detected in water samples from CTC, DHW, CW, and DF. Regarding the geographic distribution, positive samples of Legionella were obtained in 14 of the 18 Spanish locations analyzed. Legionella non-pneumophila was the most prevalent and was isolated from water samples from 13 different geographical locations (72%). Legionella anisa and Legionella jordanis were the most frequently non-pneumophila species isolated. Legionella donaldsonii was isolated for the first time in the water distribution networks in Spain. Legionella pneumophila sg 2-14 was detected in 13 locations and Legionella pneumophila sg 1 in 11 locations. Therefore, our study concludes that the presence of Legionella pneumophila and Legionella non-pneumophila species in these systems can be a potential threat to public health and should be examined thoroughly with complementary techniques, such as molecular techniques as a screen for routine diagnosis.

Legionella feeleii: Ubiquitous Pathogen in the Environment and Causative Agent of Pneumonia.

L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires' disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors (age, insulin-dependent diabetes, and heart disease). The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis.

Phylogenetic approach to the variability of the microsporidian Enterocytozoon bieneusi and its implications for inter- and intrahost transmission.

Enterocytozoon bieneusi is a microsporidian parasite that infects many vertebrate animals, including humans. The rDNA internal transcribed spacer (ITS) shows a hypervariable sequence; however, so far no clear information has been inferred about strain evolution in this species. We reviewed all the sequences described and performed a phylogenetic study. Four groups of sequences strongly differentiated from each other were detected, although most of the isolates (94%) corresponded to group I. The highly diverse sequences of this group were analyzed using median-joining networks. The host species (humans, pets, swine, cattle, birds, and wild animals) and the continents of origin of the isolates were considered. Central haplotypes in the network were obtained from very diverse hosts and geographical origins. The results show that although E. bieneusi has a broad host specificity, transmission is not completely free: some strains were able to circulate within a given host species and were only occasionally transmitted to another host. Additionally, while not relevant for swine or cattle hosts, geography seems to be a relevant factor for human infection by E. bieneusi.

Zoonotic Microsporidia in Wild Lagomorphs in Southern Spain.

Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia (Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi) in organ meats of European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits (0.8%; 95% CI: 0.0-1.7%) but not in hares (0.0%; 95% CI: 0.0-4.6%), whereas E. intestinalis DNA was found in one wild rabbit (0.3%; 95% CI: 0.0-0.8%) and three Iberian hares (3.8%; 95% CI: 0.0-8.0%). Neither E. hellem nor E. cuniculi infection were detected in the 462 (0.0%; 95% CI: 0.0-0.8%) lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors' knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.

Presence and interaction of free-living amoebae and amoeba-resisting bacteria in water from drinking water treatment plants.

Free-living amoebae (FLA) are ubiquitous and many isolates have been shown to be infected with amoeba-resisting bacteria, as the example of Acanthamoeba and Legionella interaction. Due to the high environmental prevalence of Acanthamoeba. in the Castilian Plateau (Spain), the aims of this work were to investigate the occurrence of Acanthamoeba and other FLA in water from several sampling points from four Drinking Water Treatment Plants (DWTP) and to investigate the presence of Legionella spp. and other amoeba-resisting bacteria in biofilms in raw and finished water, taking into account that no legislation exists for this protozoa control. Acanthamoeba was detected at different sampling points, and sand filters seemed to contribute to amoebic enrichment. After ozonation, a temporary decrease in viable amoebae was observed. The genotypes detected were T3, T4, and T5, revealing the first report of genotype T5 in waters from this region. Moreover, Balamuthia mandrillaris, Vermamoeba vermiformis and Paravahlkampfia sp. were detected. Regarding Legionella, PCR detection in raw and finished water was higher than by agar culture, but even higher after Acanthamoeba co-culture. Also, Legionella's presence was higher in raw water than in finished water. The decrease of free Legionella observed from raw (27.5%, by PCR) to finished water (3.4% by PCR) contrasted with the increase of Legionella-infected FLA from raw (30.7%) to finished water (52%). At biofilm, free Legionella was not detected, and the percentage of infected FLA was low (3.8%). Legionella species identified in these samples were L. drozanskii, L. donaldsonii and L. feeleii. Additionally, Acanthamoeba co-culture led to the isolation of Pseudomonas aeruginosa, P. stutzeri, P. fluorecens, Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The highly disseminated presence of Acanthamoeba and the detection of amoeba-resisting bacteria inside amoebae highlight the importance of developing methods for controlling FLA in order to limit human pathogenic amoeba-resisting bacteria survival to the water purification processes.

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs.

Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.

Experimental toxocariosis in BALB/c mice: effect of the inoculation dose on brain and eye involvement.

Ocular toxocarosis (OT) is a well-established disease. However, our understanding of the migratory route, time and circumstances that produce ocular invasion are not clear. To improve our knowledge of factors related to ocular invasion, BALB/c mice were inoculated with simple doses (SD) of 6, 12, 50, 100, 200 and 1000 embryonated eggs (EE) and multiple doses (MD) of 200 and 1000 EE. Brains and eyes were studied for the presence of larvae in animals sacrificed on days 3, 5, 7, 9, 11, 13, 15, 17, 19, 40, 80 and 120 in SD and on days 28, 30, 32, 34, 36, 38, 40, 42, 46, 87 and 127 in MD. The humoral immune responses were studied by ELISA using excretory-secretory antigen. Due to the considerable number of days tested, results showed are based on one set of experiments. However, each point studied represents the result obtained from a group of five mice. We have shown that the eye involvement with Toxocara canis larvae is a phenomenon mainly produced once larvae have reached the brain. There is a direct relationship between the parasitic load and the number of ocular larvae. Moreover, the arrival of larvae to the eye is an independent event, unrelated to the kind of administered dose (SD, MD), although the number of the brain larvae was higher in the cases of MD. High levels of specific antibodies were observed but they did not prevent the arrival of the larvae to the brain and the eye.