Equine Rhinitis A Virus Mutants with Altered Acid Resistance Unveil a Key Role of VP3 and Intrasubunit Interactions in the Control of the pH Stability of the Aphthovirus Capsid.

Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and is genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intraprotomer interface between VP3 and VP2 or between VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV. IMPORTANCE: The viral capsid constitutes a sort of dynamic nanomachine that protects the viral genome against environmental assaults while accomplishing important functions such as receptor attachment for viral entry or genome release. We have explored the molecular determinants of aphthovirus capsid stability by isolating and characterizing a panel of equine rhinitis A virus mutants that differed on their acid sensitivity. All the mutations were located within a specific region of the capsid, the intraprotomer interface among capsid proteins, thus providing new insights into the regions that control the acid stability of aphthovirus capsid. These findings could positively contribute to the development of antiviral approaches targeting aphthovirus uncoating or the refinement of vaccine strategies based on capsid stabilization.

Modification of the Host Cell Lipid Metabolism Induced by Hypolipidemic Drugs Targeting the Acetyl Coenzyme A Carboxylase Impairs West Nile Virus Replication.

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bite of mosquitoes that causes meningitis and encephalitis in humans, horses, and birds. Several studies have highlighted that flavivirus infection is highly dependent on cellular lipids for virus replication and infectious particle biogenesis. The first steps of lipid synthesis involve the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA that is catalyzed by the acetyl-CoA carboxylase (ACC). This makes ACC a key enzyme of lipid synthesis that is currently being evaluated as a therapeutic target for different disorders, including cancers, obesity, diabetes, and viral infections. We have analyzed the effect of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) on infection by WNV. Lipidomic analysis of TOFA-treated cells confirmed that this drug reduced the cellular content of multiple lipids, including those directly implicated in the flavivirus life cycle (glycerophospholipids, sphingolipids, and cholesterol). Treatment with TOFA significantly inhibited the multiplication of WNV in a dose-dependent manner. Further analysis of the antiviral effect of this drug showed that the inhibitory effect was related to a reduction of viral replication. Furthermore, treatment with another ACC inhibitor, 3,3,14,14-tetramethylhexadecanedioic acid (MEDICA 16), also inhibited WNV infection. Interestingly, TOFA and MEDICA 16 also reduced the multiplication of Usutu virus (USUV), a WNV-related flavivirus. These results point to the ACC as a druggable cellular target suitable for antiviral development against WNV and other flaviviruses.

The pH Stability of Foot-and-Mouth Disease Virus Particles Is Modulated by Residues Located at the Pentameric Interface and in the N Terminus of VP1.

UNLABELLED: The picornavirus foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects important livestock species. The FMDV capsid is highly acid labile, and viral particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid sensitivity is related to the mechanism of viral uncoating and genome penetration from endosomes. In this study, we have analyzed the molecular basis of FMDV acid-induced disassembly by isolating and characterizing a panel of novel FMDV mutants differing in acid sensitivity. Amino acid replacements altering virion stability were preferentially distributed in two different regions of the capsid: the N terminus of VP1 and the pentameric interface. Even more, the acid labile phenotype induced by a mutation located at the pentameric interface in VP3 could be compensated by introduction of an amino acid substitution in the N terminus of VP1. These results indicate that the acid sensitivity of FMDV can be considered a multifactorial trait and that virion stability is the fine-tuned product of the interaction between residues from different capsid proteins, in particular those located within the N terminus of VP1 or close to the pentameric interface. IMPORTANCE: The viral capsid protects the viral genome from environmental factors and contributes to virus dissemination and infection. Thus, understanding of the molecular mechanisms that modulate capsid stability is of interest for the basic knowledge of the biology of viruses and as a tool to improve the stability of conventional vaccines based on inactivated virions or empty capsids. Using foot-and-mouth disease virus (FMDV), which displays a capsid with extreme acid sensitivity, we have performed a genetic study to identify the molecular determinants involved in capsid stability. A panel of FMDV mutants with differential sensitivity to acidic pH was generated and characterized, and the results showed that two different regions of FMDV capsid contribute to modulating viral particle stability. These results provide new insights into the molecular mechanisms of acid-mediated FMDV uncoating.

An increase in acid resistance of foot-and-mouth disease virus capsid is mediated by a tyrosine replacement of the VP2 histidine previously associated with VP0 cleavage.

The foot-and-mouth disease virus (FMDV) capsid is highly acid labile, but introduction of amino acid replacements, including an N17D change in VP1, can increase its acid resistance. Using mutant VP1 N17D as a starting point, we isolated a virus with higher acid resistance carrying an additional replacement, VP2 H145Y, in a residue highly conserved among picornaviruses, which has been proposed to be responsible for VP0 cleavage. This mutant provides an example of the multifunctionality of picornavirus capsid residues.

Preparation of Highly Stable and Cost-Efficient Antiviral Materials for Reducing Infections and Avoiding the Transmission of Viruses such as SARS-CoV-2.

The current global pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has demonstrated the necessity to develop novel materials with antimicrobial and antiviral activities to prevent the infection. One significant route for the spread of diseases is by the transmission of the virus through contact with contaminated surfaces. Antiviral surface treatments can help to reduce or even avoid these hazards. In particular, the development of active-virucidal fabrics or paints represents a very important challenge with multiple applications in hospitals, public transports, or schools. Modern, cutting-edge methods for creating antiviral surface coatings use either materials with a metal base or sophisticated synthetic polymers. Even if these methods are effective, they will still face significant obstacles in terms of large-scale applicability. Here, we describe the preparation of fabrics and paints treated with a scaled-up novel nanostructured biohybrid material composed of very small crystalline phosphate copper(II) nanoparticles, synthesized based on a technology that employs the use of a small amount of biological agent for its formation at room temperature in aqueous media. We demonstrate the efficient inactivation of the human coronavirus 229E (HCoV-229E), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and non-enveloped human rhinovirus 14 (HRV-14) (>99.9%) using an inexpensive, ecologically friendly coating agent. The reactive oxygen species produced during the oxidation of water or the more intensive reaction with hydrogen peroxide are believed to be the cause of the antiviral mechanism of the nanostructured material. In contrast to the release of a specific antiviral drug, this process does not consume the surface coating and does not need regeneration. A 12-month aging research that revealed no decline in antiviral activity is proof that the coating is durable in ambient circumstances. Also, the coated fabric can be reused after different washing cycles, even at moderate to high temperatures.

A comparative study of serological tests used in the diagnosis of Toxoplasma gondii infection in small ruminants evidenced the importance of cross-reactions for harmonizing diagnostic performance.

Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results.

Clear polyurethane coatings with excellent virucidal properties: Preparation, characterization and rapid inactivation of human coronaviruses 229E and SARS-CoV-2.

Commercial polyurethane (PU) coating formulations have been modified with 1-(hydroxymethyl)-5,5-dimethylhydantoin (HMD) both in bulk (0.5 and 1% w/w) and onto the coatings surface as an N-halamine precursor, to obtain clear coatings with high virucidal activity. Upon immersion in diluted chlorine bleaching, the hydantoin structure on the grafted PU membranes was transformed into N-halamine groups, with a high surface chlorine concentration (40-43µg/cm2). Fourier transform infrared spectroscopy (FTIR) spectroscopy, thermogravimetric analysis (TGA), energy-dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS) and iodometric titration were used to characterize the coatings and quantify the chlorine contents of the PU membranes after chlorination. Biological evaluation of their activity against Staphylococcus aureus (Gram-positive bacteria) and human coronaviruses HCoV-229E and SARS-CoV-2 was performed, and high inactivation of these pathogens was observed after short contact times. The inactivation of HCoV-229E was higher than 98% for all modified samples after just 30 minutes, whereas it was necessary 12 hours of contact time for complete inactivation of SARS-CoV-2. The coatings were fully rechargeable by immersion in diluted chlorine bleach (2% v/v) for at least 5 chlorination-dechlorination cycles. Moreover, the performance of the antivirus efficiency of the coatings is considered as long-lasting, because experiments of reinfection of the coatings with HCoV-229E coronavirus did not show any loss of the virucidal activity after three consecutive infection cycles without reactivation of the N-halamine groups.

Modulation of foot-and-mouth disease virus pH threshold for uncoating correlates with differential sensitivity to inhibition of cellular Rab GTPases and decreases infectivity in vivo.

The role of cellular Rab GTPases that govern traffic between different endosome populations was analysed on foot-and-mouth disease virus (FMDV) infection. Changes of viral receptor specificity did not alter Rab5 requirement for infection. However, a correlation between uncoating pH and requirement of Rab5 for infection was observed. A mutant FMDV with less acidic uncoating pH threshold was less sensitive to inhibition of Rab5, whereas another mutant with more acidic requirements was more sensitive to inhibition of Rab5. On the contrary, opposed correlations between uncoating pH and dependence of Rab function were observed upon expression of dominant-negative forms of Rab7 or 11. Modulation of uncoating pH also reduced FMDV virulence in suckling mice. These results are consistent with FMDV uncoating inside early endosomes and indicate that displacements from optimum pH for uncoating reduce viral fitness in vivo.

Inhibition of enveloped virus infection of cultured cells by valproic acid.

Valproic acid (VPA) is a short-chain fatty acid commonly used for treatment of neurological disorders. As VPA can interfere with cellular lipid metabolism, its effect on the infection of cultured cells by viruses of seven viral families relevant to human and animal health, including eight enveloped and four nonenveloped viruses, was analyzed. VPA drastically inhibited multiplication of all the enveloped viruses tested, including the zoonotic lymphocytic choriomeningitis virus and West Nile virus (WNV), while it did not affect infection by the nonenveloped viruses assayed. VPA reduced vesicular stomatitis virus infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.

Assessment of Surface Disinfection Effectiveness of Decontamination System COUNTERFOG® SDR-F05A+ Against Bacteriophage ɸ29.

The experience of COVID19 pandemic has demonstrated the real concern of biological agents dispersed in the air and surfaces environments. Therefore, the need of a fast and large-scale disinfection method has arisen for prevention of contagion. COUNTERFOG® is an innovative technology developed for large-scale decontamination of air and surfaces. The objective of this study is to assess experimentally the effectiveness of COUNTERFOG® in disinfecting viral-contaminated surfaces. We also aim to measure the necessary time to disinfect said surfaces. Stainless steel surfaces were contaminated with bacteriophage φ29 and disinfected using COUNTERFOG® SDR-F05A+, which uses a sodium hypochlorite solution at different concentrations and for different exposure times. A log reduction over 6 logs of virus titer is obtained in 1 min with 1.2% sodium hypochlorite when the application is direct; while at a radial distance of 5 cm from the point of application the disinfection reaches a reduction of 5.5 logs in 8 min. In the same way, a higher dilution of the sodium hypochlorite concentration (0.7% NaOCl) requires more exposure time (16 min) to obtain the same log reduction (> 6 logs). COUNTERFOG® creates, in a short time and at a distance of 2 m from the point of application, a thin layer of disinfectant that covers the surfaces. The selection of the concentration and exposure time is critical for the efficacy of disinfection. These tests demonstrate that a concentration between 0.7- 1.2% sodium hypochlorite is enough for a fast and efficient ɸ29 phage inactivation. The fact that ɸ29 phage is more resistant to disinfection than SARS-CoV-2 sustains this disinfection procedure.

Fast Air-to-Liquid Sampler Detects Surges in SARS-CoV-2 Aerosol Levels in Hospital Rooms.

The COVID-19 pandemic highlighted the dangers of airborne pathogen transmission. SARS-CoV-2 is known to be transmitted through aerosols; however, little is known about the dynamics of these aerosols in real environments, the conditions, and the minimum viral load required for infection. Efficiently measuring and capturing pathogens present in the air would help to understand the infection process. Air samplers usually take several hours to obtain an air sample. In this work a fast (1-2 min) method for capturing bioaerosols into a liquid medium has been tested in hospital rooms with COVID-19 patients. This fast sampling allows detecting transient levels of aerosols in the air. SARS-CoV-2 RNA is detected in aerosols from several hospital rooms at different levels. Interestingly, there are sudden boosts of the SARS-CoV-2 load in the air, suggesting that SARS-CoV-2 could be released abundantly at certain moments. These results show that the distribution of SARS-CoV-2-containing aerosols is not homogeneous in the hospital room. This technology is a fast and effective tool for capturing airborne matter in a very short time, which allows for fast decision-making any kind of hazard in the air is detected. It is also useful for a better understanding of aerosols dynamics.

Adaptive value of foot-and-mouth disease virus capsid substitutions with opposite effects on particle acid stability.

Foot-and-mouth disease virus (FMDV) is a picornavirus that exhibits an extremely acid sensitive capsid. This acid lability is directly related to its mechanism of uncoating triggered by acidification inside cellular endosomes. Using a collection of FMDV mutants we have systematically analyzed the relationship between acid stability and the requirement for acidic endosomes using ammonium chloride (NH4Cl), an inhibitor of endosome acidification. A FMDV mutant carrying two substitutions with opposite effects on acid-stability (VP3 A116V that reduces acid stability, and VP1 N17D that increases acid stability) displayed a rapid shift towards acid lability that resulted in increased resistance to NH4Cl as well as to concanamicyn A, a different lysosomotropic agent. This resistance could be explained by a higher ability of the mutant populations to produce NH4Cl-resistant variants, as supported by their tendency to accumulate mutations related to NH4Cl-resistance that was higher than that of the WT populations. Competition experiments also indicated that the combination of both amino acid substitutions promoted an increase of viral fitness that likely contributed to NH4Cl resistance. This study provides novel evidences supporting that the combination of mutations in a viral capsid can result in compensatory effects that lead to fitness gain, and facilitate space to an inhibitor of acid-dependent uncoating. Thus, although drug-resistant variants usually exhibit a reduction in viral fitness, our results indicate that compensatory mutations that restore this reduction in fitness can promote emergence of resistance mutants.

Development and validation of a time-resolved fluorescence immunoassay for the detection of anti-Toxoplasma gondii antibodies in goats.

Toxoplasma gondii is a worldwide distributed parasite causing abortions and fetal malformations in small ruminants. The aim of this study was to design and validate a new immunoassay based on the use of TgSAG1-GRA8 chimeric antigen for the detection of anti-T. gondii antibodies in serum of goats. First, a time-resolved fluorescence immunoassay (TgSAG1-GRA8-TRFIA) was developed. In addition, the diagnostic performance of TgSAG1-GRA8-TRFIA was compared with an optimized enzyme-linked immunosorbent assay (TgSALUVET-ELISA) and a Western Blot (WB), both based on whole T. gondii tachyzoite antigenic extract. The TgSAG1-GRA8-TRFIA has shown a high intra- and inter-assay precision, analytical sensitivity and accuracy. The ROC analysis of this assay showed an optimal cut-off of 217.4 Units of Fluorometry for T. gondii (UFT), with 92 % of sensitivity and 90.48 % of specificity. A positive and statistically significant Spearman's correlation with TgSALUVET-ELISA was detected, and kappa value was 0.83, presenting high agreement with both methods. However, TgSAG1-GRA8 protein showed cross-reactivity with specific anti-Neospora caninum antibodies. Thus, TgSAG-1-GRA8 chimeric antigen seems not to be an ideal option for the serodiagnosis of T. gondii infection in goats unless combined with the serodiagnosis of N. caninum infection in parallel. In the light of the results obtained, a comprehensive study on the existence of cross-reactivities between T. gondii antigens used in serological tests employed in animal health and specific antibodies directed against Toxoplasmatinae parasites should be performed.

Dynamics of Neospora caninum-Associated Abortions in a Dairy Sheep Flock and Results of a Test-and-Cull Control Programme.

Neospora caninum is an apicomplexan parasite that can cause abortions and perinatal mortality in sheep. Although ovine neosporosis has been described worldwide, there is a lack of information about the relationship between N. caninum serostatus and the reproductive performance. In this study, we described the infection dynamics in a dairy sheep flock with an abortion rate up to 25% and a N. caninum seroprevalence of 32%. Abortions were recorded in 36% and 9% of seropositive and seronegative sheep, respectively. Seropositive sheep were more likely to abort twice (OR = 4.44) or three or more times (OR = 10.13) than seronegative sheep. Endogenous transplacental transmission was the main route of transmission since 86% of seropositive sheep had seropositive offspring. Within dams that had any abortion, seropositive sheep were more likely than seronegative ones to have female descendants that aborted (OR = 8.12). The slight increase in seropositivity with the age, the low percentage of animals with postnatal seroconversion or with low avidity antibodies, and the seropositivity of one flock dog, indicated that horizontal transmission might have some relevance in this flock. A control programme based on selective culling of seropositive sheep and replacement with seronegative animals was effective in reducing the abortion rate to 7.2%.

Internalization of swine vesicular disease virus into cultured cells: a comparative study with foot-and-mouth disease virus.

We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses.

Clinical Infections by Herpesviruses in Patients Treated with Valproic Acid: A Nested Case-Control Study in the Spanish Primary Care Database, BIFAP.

The objective of this study is to evaluate the risk of clinical infections by herpesviruses in patients exposed to valproic acid (VPA). We performed a case-control study nested in a primary cohort selected from the Spanish primary care population-based research database BIFAP (Base de datos para la Investigación Farmacoepidemiológica en Atención Primaria) over the period 2001-2015. The events of interest were those diseases caused by any herpesviruses known to infect humans. For each case, up to 10 controls per case matched by age, gender, and calendar date were randomly selected. A conditional logistic regression was used to compute adjusted odds ratios (OR) and their 95% confidence intervals (95% CI). Current use of VPA was associated with a trend towards a reduced risk of clinical infections by herpesviruses as compared with non-users (OR 0.84; CI 95% 0.7-1.0; p = 0.057). Among current users, a trend to a decreased risk with treatment durations longer than 90 days was also observed. The results show a trend to a reduced risk of clinical infection by herpesviruses in patients exposed to VPA. These results are consistent with those in vitro studies showing that, in cultured cells, VPA can inhibit the production of the infectious progeny of herpesviruses. This study also shows the efficient use of electronic healthcare records for clinical exploratory research studies.

A time-resolved fluorescence immunoassay for the detection of anti-Neospora caninum antibodies in sheep.

Neospora caninum is a protozoan parasite (Phylum Apicomplexa) that has been recently suggested as a relevant cause of reproductive disorders in small ruminants. The aim of the present study is to develop and validate a new serological test based on time resolved fluorescency using N. caninum GRA7 recombinant antigen (GRA7-TRFIA) for the detection of N. caninum antibodies in sheep. A total of 346 serum samples (208 from experimentally infected sheep, 117 from a dairy farm with a previous history of Neospora-associated abortion, and 21 negative sera) were used. The validation of the new assay was performed by the evaluation of assay precision, analytical sensitivity (Se), accuracy and cross reactivity. In the experimentally infected sheep, antibody kinetics was compared between GRA7-TRFIA and an in house N. caninum tachyzoite soluble extract-based ELISA (NcSALUVET ELISA) by Wilcoxon matched-pairs signed rank test. The cut-off and diagnostic Se and specificity (Sp) of GRA7-TRFIA was estimated by ROC analysis with field samples. In addition, concordance and correlation between GRA7-TRFIA and a commercial ELISA and NcSALUVET ELISA were assessed by kappa value and Spearman correlation coefficient, respectively. Overall, GRA7-TRFIA showed an adequate precision, analytical Se and accuracy to detect anti-N. caninum antibodies in ovine serum, and no cross reactivity with the closely related protozoan Toxoplasma gondii. In naturally infected sheep, 100% Se and 95.35% Sp were obtained for a cut-off point of 62.68 Units of Fluorometry for N. caninum (UFN). Moreover, GRA7-TRFIA allowed earlier detection of N. caninum infection than NcSALUVET ELISA in experimentally infected sheep.

Pharmacological Inhibition of Protein Kinase C Reduces West Nile Virus Replication.

Flaviviruses are relevant animal and human pathogens that include West Nile virus (WNV), Japanese encephalitis virus, dengue virus, or Zika virus, among others. Currently, no licensed therapy is available to fight flaviviral infections. Protein kinases C (PKCs) constitute a family of multifunctional lipid-dependent isoenzymes that regulate a wide variety of cellular processes (apoptosis, differentiation, proliferation, cellular transformation, motility, adhesion, etc.) being currently considered at the front line of drug development for the treatment of diverse human disorders. PKCs have also been implicated in different steps during viral replication; however, nowadays, results regarding their role in flavivirus replication are controversial. Here we demonstrate that calphostin C and chelerythrine, two broad-PKC inhibitors that target conventional, novel and atypical PKCs, significantly inhibit WNV multiplication in cell culture without affecting cell viability. A reduction of viral yields was observed in treated cells when compared with mock-treated cells. Likewise, immunofluorescence detection of viral enveloped E protein was reduced in treated cells, as was the amount of viral RNA released to the supernatant, mainly in those treated with chelerythrine. On the other hand, two PKC inhibitors specific for conventional and novel isoforms (staurosporine and enzastaurine) did not show any significant effect in WNV multiplication. These results suggested that PKCs, more probably atypical PKCs, are likely involved in WNV multiplication, although both broad-spectrum tested drugs seem to act through different mechanisms, and point to them as potential antiviral candidates for WNV, as well as for other related flaviviruses.

Preserved immunogenicity of an inactivated vaccine based on foot-and-mouth disease virus particles with improved stability.

Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects important livestock species. Vaccines based on inactivated FMDV virions provide a useful tool for the control of this pathogen. However, long term storage at 4°C (the temperature for vaccine storage) or ruptures of the cold chain, provoke the dissociation of virions, reducing the immunogenicity of the vaccine. An FMDV mutant carrying amino acid replacements VP1 N17D and VP2 H145Y isolated previously rendered virions with increased resistance to dissociation at 4°C. We have evaluated the immunogenicity in swine (a natural FMDV host) of a chemically inactivated vaccine based on this mutant. The presence of these amino acid substitutions did not compromise the immunological potential, including its ability to elicit neutralizing antibodies. These results support the feasibility of this kind of mutants with increased capsid stability as suitable viruses for producing improved FMDV vaccines.