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Flaviviruses affect the lives of millions of people in endemic regions and also have the potential to impact non-endemic areas. Factors such as climate change, global warming, deforestation, and increased travel and trade are linked to the spread of flaviviruses into new habitats and host species. Given the absence of specific treatments and the limited availability of vaccines, it is imperative to understand the biology of flaviviruses and develop rapid and sensitive diagnostic tests. These measures are essential for preventing the transmission of these potentially life-threatening pathogens. Flavivirus infections are mainly diagnosed using conventional methods. However, these techniques present several drawbacks, including high expenses, time-consuming procedures, and the need for skilled professionals. The search for fast, easy-to-use, and affordable alternative techniques as a feasible solution for developing countries is leading to the search for new methods in the diagnosis of flaviviruses, such as biosensors. This review provides a comprehensive overview of different biosensor detection strategies for flaviviruses and describes recent advances in diagnostic technologies. Finally, we explore their future prospects and potential applications in pathogen detection. This review serves as a valuable resource to understand advances in ongoing research into new biosensor-based diagnostic methods for flaviviruses., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The emergence of SARS-CoV-2 in 2019 and its rapid spread throughout the world has caused the largest pandemic of our modern era. The zoonotic origin of this pathogen highlights the importance of the One Health concept and the need for a coordinated response to this kind of threats. Since its emergence, the virus has caused >7 million deaths worldwide. However, the animal source for human outbreaks remains unknown. The ability of the virus to jump between hosts is facilitated by the presence of the virus receptor, the highly conserved angiotensin-converting enzyme 2 (ACE2), found in various mammals. Positivity for SARS-CoV-2 has been reported in various species, including domestic animals and livestock, but their potential role in bridging viral transmission to humans is still unknown. Additionally, the virus has evolved over the pandemic, resulting in variants with different impacts on human health. Therefore, suitable animal models are crucial to evaluate the susceptibility of different mammalian species to this pathogen and the adaptability of different variants. In this work, we established a transgenic mouse model that expresses the feline ACE2 protein receptor (cACE2) under the human cytokeratin 18 (K18) gene promoter's control, enabling high expression in epithelial cells, which the virus targets. Using this model, we assessed the susceptibility, pathogenicity, and transmission of SARS-CoV-2 variants. Our results show that the sole expression of the cACE2 receptor in these mice makes them susceptible to SARS-CoV-2 variants from the initial pandemic wave but does not enhance susceptibility to omicron variants. Furthermore, we demonstrated efficient contact transmission of SARS-CoV-2 between transgenic mice that express either the feline or the human ACE2 receptor., Competing Interests: The authors report there are no competing interests to declare., (© 2024 The Authors. Published by Elsevier B.V.)

Integration of thin-film oxide piezoelectrics on glass is imperative for the next generation of transparent electronics to attain sensing and actuating functions. However, their crystallization temperature (above 650 °C) is incompatible with most glasses. We developed a flash lamp process for the growth of piezoelectric lead zirconate titanate films. The process enables crystallization on various types of glasses in a few seconds only. The functional properties of these films are comparable to the films processed with standard rapid thermal annealing at 700 °C. A surface haptic device was fabricated with a 1 μm-thick film (piezoelectric e 33,f of -5 C m -2 ). Its ultrasonic surface deflection reached 1.5 μm at 60 V, sufficient for its use in surface rendering applications. This flash lamp annealing process is compatible with large glass sheets and roll-to-roll processing and has the potential to significantly expand the applications of piezoelectric devices on glass., (© 2024. The Author(s).)

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of West Nile disease (WND) can curse with meningitis, encephalitis or acute flaccid paralysis. A better understanding of the physiopathology associated with disease progression is mandatory to find biomarkers and effective therapies. In this scenario, blood derivatives (plasma and serum) constitute the more commonly used biofluids due to its ease of collection and high value for diagnostic purposes. Therefore, the potential impact of this virus in the circulating lipidome was addressed combining the analysis of samples from experimentally infected mice and naturally WND patients. Our results unveil dynamic alterations in the lipidome that define specific metabolic fingerprints of different infection stages. Concomitant with neuroinvasion in mice, the lipid landscape was dominated by a metabolic reprograming that resulted in significant elevations of circulating sphingolipids (ceramides, dihydroceramides, and dihydrosphingomyelins), phosphatidylethanolamines and triacylglycerols. Remarkably, patients suffering from WND also displayed an elevation of ceramides, dihydroceramides, lactosylceramides, and monoacylglycerols in their sera. The dysregulation of sphingolipid metabolism by WNV may provide new therapeutic opportunities and supports the potential of certain lipids as novel peripheral biomarkers of WND progression.

Background: Viral rewiring of host bioenergetics and immunometabolism may provide novel targets for therapeutic interventions against viral infections. Here, we have explored the effect on bioenergetics during the infection with the mosquito-borne flavivirus West Nile virus (WNV), a medically relevant neurotropic pathogen causing outbreaks of meningitis and encephalitis worldwide., Results: A systematic literature search and meta-analysis pointed to a misbalance of glucose homeostasis in the central nervous system of WNV patients. Real-time bioenergetic analyses confirmed upregulation of aerobic glycolysis and a reduction of mitochondrial oxidative phosphorylation during viral replication in cultured cells. Transcriptomics analyses in neural tissues from experimentally infected mice unveiled a glycolytic shift including the upregulation of hexokinases 2 and 3 (Hk2 and Hk3) and pyruvate dehydrogenase kinase 4 (Pdk4). Treatment of infected mice with the Hk inhibitor, 2-deoxy-D-glucose, or the Pdk4 inhibitor, dichloroacetate, alleviated WNV-induced neuroinflammation., Conclusions: These results highlight the importance of host energetic metabolism and specifically glycolysis in WNV infection in vivo. This study provides proof of concept for the druggability of the glycolytic pathway for the future development of therapies to combat WNV pathology., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The flavivirus life cycle is strictly dependent on cellular lipid metabolism. Polyphenols like gallic acid and its derivatives are promising lead compounds for new therapeutic agents as they can exert multiple pharmacological activities, including the alteration of lipid metabolism. The evaluation of our collection of polyphenols against West Nile virus (WNV), a representative medically relevant flavivirus, led to the identification of N , N '-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its 2,3,4-trihydroxybenzamide regioisomer as selective antivirals with low cytotoxicity and high antiviral activity (half-maximal effective concentrations [EC 50 s] of 2.2 and 0.24 μM, respectively, in Vero cells; EC 50 s of 2.2 and 1.9 μM, respectively, in SH-SY5Y cells). These polyphenols also inhibited the multiplication of other flaviviruses, namely, Usutu, dengue, and Zika viruses, exhibiting lower antiviral or negligible antiviral activity against other RNA viruses. The mechanism underlying their antiviral activity against WNV involved the alteration of sphingolipid metabolism. These compounds inhibited ceramide desaturase (Des1), promoting the accumulation of dihydrosphingomyelin (dhSM), a minor component of cellular sphingolipids with important roles in membrane properties. The addition of exogenous dhSM or Des1 blockage by using the reference inhibitor GT-11 { N -[(1 R ,2 S )-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide} confirmed the involvement of this pathway in WNV infection. These results unveil the potential of novel antiviral strategies based on the modulation of the cellular levels of dhSM and Des1 activity for the control of flavivirus infection.

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 IC 50 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 EC 50 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Flavivirus comprises globally emerging and re-emerging pathogens such as Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), among others. Although some vaccines are available, there is an unmet medical need as no effective antiviral treatment has been approved for flaviviral infections. The development of host-directed antivirals (HDAs) targeting host factors that are essential for viral replication cycle offers the opportunity for the development of broad-spectrum antivirals. In the case of flaviviruses, recent studies have revealed that neutral sphingomyelinase 2, (nSMase2), involved in lipid metabolism, plays a key role in WNV and ZIKV infection. As a proof of concept, we have determined the antiviral activity of the non-competitive nSMase2 inhibitor DPTIP against WNV and ZIKV virus. DPTIP showed potent antiviral activity with EC 50 values of 0.26 µM and 1.56 µM for WNV and ZIKV, respectively. In order to unravel the allosteric binding site of DPTIP in nSMase2 and the details of the interaction, computational studies have been carried out. These studies have revealed that DPTIP could block the DK switch in nSMase2. Moreover, the analysis of the residues contributing to the binding identified His463 as a crucial residue. Interestingly, the inhibitory activity of DPTIP on the H463A mutant protein supported our hypothesis. Thus, an allosteric cavity in nSMase2 has been identified that can be exploited for the development of new inhibitors with anti-flaviviral activity., Competing Interests: The authors declare no conflict of interest.

Zika virus (ZIKV) is a mosquito-borne flavivirus whose infection in pregnant women is associated with a spectrum of birth defects, which are together referred as Congenital Zika Syndrome. In addition, ZIKV can also induce Guillain-Barré syndrome, which is an autoimmune disease with neurological symptoms. The recent description of the first local infections of ZIKV in the European continent together with the expansion of one of its potential vectors, the Asian tiger mosquito (Aedes albopictus) , invite us to be prepared for future outbreaks of ZIKV in this geographical region. However, the antigenic similarities of ZIKV with other flaviviruses can lead to an immune cross-reactivity with other circulating flaviviruses inducing, in some cases, flavivirus-disease exacerbation by antibody-dependent enhancement (ADE) of infection, which is a major concern for ZIKV vaccine development. Until now, West Nile virus (WNV) is the main medically relevant flavivirus circulating in the Mediterranean Basin. Therefore, anticipating the potential scenario of emergency vaccination against ZIKV in areas of Europe where WNV is endemic, in this investigation, we have evaluated the cross-reactivity between WNV and our previously developed ZIKV vaccine candidate based on modified vaccinia virus Ankara (MVA) vector expressing ZIKV structural proteins (MVA-ZIKV). To this end, mice were first immunized with MVA-ZIKV, subsequently challenged with WNV, and then, the ZIKV- and WNV-specific immune responses and protection against WNV were evaluated. Our results indicate low cross-reactivity between the MVA-ZIKV vaccine candidate and WNV and absence of ADE, supporting the safety of this ZIKV vaccine candidate in areas where the circulation of WNV is endemic.

West Nile virus (WNV) is a flavivirus which transmission cycle is maintained between mosquitoes and birds, although it occasionally causes sporadic outbreaks in horses and humans that can result in serious diseases and even death. Since its first isolation in Africa in 1937, WNV had been considered a neglected pathogen until its recent spread throughout Europe and the colonization of America, regions where it continues to cause outbreaks with severe neurological consequences in humans and horses. Although our knowledge about the characteristics and consequences of the virus has increased enormously lately, many questions remain to be resolved. Here, we thoroughly update our knowledge of different aspects of the WNV life cycle: virology and molecular classification, host cell interactions, transmission dynamics, host range, epidemiology and surveillance, immune response, clinical presentations, pathogenesis, diagnosis, prophylaxis (antivirals and vaccines), and prevention, and we highlight those aspects that are still unknown and that undoubtedly require further investigation.

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile, Japanese encephalitis, Zika, and dengue viruses. USUV emerged in 1996 in Europe, where quickly spread across the continent causing a considerable number of bird deaths and varied neurological disorders in humans, including encephalitis, meningoencephalitis, or facial paralysis, thus warning about USUV as a potential health threat. USUV replication takes place on the endoplasmic reticulum (ER) of infected cells, inducing ER stress and resulting in the activation of stress-related cellular pathways collectively known as the integrated stress response (ISR). The alpha subunit of the eukaryotic initiation factor eIF2 (eIF2α), the core factor in this pathway, is phosphorylated by stress activated kinases: protein kinase R (PKR), PKR-like endoplasmic reticulum kinase (PERK), heme-regulated inhibitor kinase (HRI), and general control non-repressed 2 kinase (GCN2). Its phosphorylation results, among others, in the downstream inhibition of translation with accumulation of discrete foci in the cytoplasm termed stress granules (SGs). Our results indicated that USUV infection evades cellular stress response impairing eIF2α phosphorylation and SGs assembly induced by treatment with the HRI activator ArsNa. This protective effect was related with oxidative stress responses in USUV-infected cells. Overall, these results provide new insights into the complex connections between the stress response and flavivirus infection in order to maintain an adequate cellular environment for viral replication., Competing Interests: The authors have declared that no competing interests exist.

Protein kinases (PKs) are enzymes that catalyze the transfer of the terminal phosphate group from ATP to a protein acceptor, mainly to serine, threonine, and tyrosine residues. PK catalyzed phosphorylation is critical to the regulation of cellular signaling pathways that affect crucial cell processes, such as growth, differentiation, and metabolism. PKs represent attractive targets for drugs against a wide spectrum of diseases, including viral infections. Two different approaches are being applied in the search for antivirals: compounds directed against viral targets (direct-acting antivirals, DAAs), or against cellular components essential for the viral life cycle (host-directed antivirals, HDAs). One of the main drawbacks of DAAs is the rapid emergence of drug-resistant viruses. In contrast, HDAs present a higher barrier to resistance development. This work reviews the use of chemicals that target cellular PKs as HDAs against virus of the Flaviviridae family ( Flavivirus and Hepacivirus ), thus being potentially valuable therapeutic targets in the control of these pathogens.

Birds are the main natural host of West Nile virus (WNV), the worldwide most distributed mosquito-borne flavivirus, but humans and equids can also be sporadic hosts. Many avian species have been reported as susceptible to WNV, particularly corvids. In the case that clinical disease develops in birds, this is due to virus invasion of different organs: liver, spleen, kidney, heart, and mainly the central nervous system, which can lead to death 24-48 h later. Nowadays, vaccines have only been licensed for use in equids; thus, the availability of avian vaccines would benefit bird populations, both domestic and wild ones. Such vaccines could be used in endangered species housed in rehabilitation and wildlife reserves, and in animals located at zoos and other recreational installations, but also in farm birds, and in those that are grown for hunting and restocking activities. Even more, controlling WNV infection in birds can also be useful to prevent its spread and limit outbreaks. So far, different commercial and experimental vaccines (inactivated, attenuated, and recombinant viruses, and subunits and DNA-based candidates) have been evaluated, with various regimens, both in domestic and wild avian species. However, there are still disadvantages that must be overcome before avian vaccination can be implemented, such as its cost-effectiveness for domestic birds since in many species the pathogenicity is low or zero, or the viability of being able to achieve collective immunity in wild birds in freedom. Here, a comprehensive review of what has been done until now in the field of avian vaccines against WNV is presented and discussed., Competing Interests: The authors declare no conflict of interest.

Flaviviruses are (re)-emerging RNA viruses strictly dependent on lipid metabolism for infection. In the search for host targeting antivirals, we explored the effect of pharmacological modulation of fatty acid metabolism during flavivirus infection. Considering the central role of acetyl-Coenzyme A carboxylase (ACC) on fatty acid metabolism, we analyzed the effect of three small-molecule ACC inhibitors (PF-05175157, PF-05206574, and PF-06256254) on the infection of medically relevant flaviviruses, namely West Nile virus (WNV), dengue virus, and Zika virus. Treatment with these compounds inhibited the multiplication of the three viruses in cultured cells. PF-05175157 induced a reduction of the viral load in serum and kidney in WNV-infected mice, unveiling its therapeutic potential for the treatment of chronic kidney disease associated with persistent WNV infection. This study constitutes a proof of concept of the reliability of ACC inhibitors to become viable antiviral candidates. These results support the repositioning of metabolic inhibitors as broad-spectrum antivirals.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that affects humans and can cause severe neurological complications, including Guillain-Barré syndrome and microcephaly. Since 2007 there have been three large outbreaks; the last and larger spread in the Americas in 2015. Actually, ZIKV is circulating in the Americas, Southeast Asia, and the Pacific Islands, and represents a potential pandemic threat. Given the rapid ZIKV dissemination and the severe neurological and teratogenic sequelae associated with ZIKV infection, the development of a safe and efficacious vaccine is critical. In this study, we have developed and characterized the immunogenicity and efficacy of a novel ZIKV vaccine based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the ZIKV prM and E structural genes (termed MVA-ZIKV). MVA-ZIKV expressed efficiently the ZIKV structural proteins, assembled in virus-like particles (VLPs) and was genetically stable upon nine passages in cell culture. Immunization of mice with MVA-ZIKV elicited antibodies that were able to neutralize ZIKV and induced potent and polyfunctional ZIKV-specific CD8 + T cell responses that were mainly of an effector memory phenotype. Moreover, a single dose of MVA-ZIKV reduced significantly the viremia in susceptible immunocompromised mice challenged with live ZIKV. These findings support the use of MVA-ZIKV as a potential vaccine against ZIKV.

Zika virus (ZIKV), a mosquito-borne flavivirus, was an almost neglected pathogen until its introduction in the Americas in 2015, where it has been responsible for a threat to global health, causing a great social and sanitary alarm due to its increased virulence, rapid spread, and an association with severe neurological and ophthalmological complications. Currently, no specific antiviral therapy against ZIKV is available, and treatments are palliative and mainly directed toward the relief of symptoms, such as fever and rash, by administering antipyretics, anti-histamines, and fluids for dehydration. Nevertheless, lately, search for antivirals has been a major aim in ZIKV investigations. To do so, screening of libraries from different sources, testing of natural compounds, and repurposing of drugs with known antiviral activity have allowed the identification of several antiviral candidates directed to both viral (structural proteins and enzymes) and cellular elements. Here, we present an updated review of current knowledge about anti-ZIKV strategies, focusing on host-directed antivirals as a realistic alternative to combat ZIKV infection.

Mosquito-borne flaviviruses are a group of RNA viruses that constitute global threats for human and animal health. Replication of these pathogens is strictly dependent on cellular lipid metabolism. We have evaluated the effect of the pharmacological activation of AMP-activated protein kinase (AMPK), a master regulator of lipid metabolism, on the infection of three medically relevant flaviviruses, namely, West Nile virus (WNV), Zika virus (ZIKV), and dengue virus (DENV). WNV is responsible for recurrent outbreaks of meningitis and encephalitis, affecting humans and horses worldwide. ZIKV has caused a recent pandemic associated with birth defects (microcephaly), reproductive disorders, and severe neurological complications (Guillain-Barré syndrome). DENV is the etiological agent of the most prevalent mosquito-borne viral disease, which can induce a potentially lethal complication called severe dengue. Our results showed, for the first time, that activation of AMPK using the specific small molecule activator PF-06409577 reduced WNV, ZIKV, and DENV infection. This antiviral effect was associated with an impairment of viral replication due to the modulation of host cell lipid metabolism exerted by the compound. These results support that the pharmacological activation of AMPK, which currently constitutes an important pharmacological target for human diseases, could also provide a feasible approach for broad-spectrum host-directed antiviral discovery., (Copyright © 2018 American Society for Microbiology.)

West Nile virus (WNV), a zoonotic pathogen naturally transmitted by mosquitoes whose natural hosts are birds, has spread worldwide during the last few decades. Resident birds play an important role in flavivirus epidemiology, since they can serve as reservoirs and facilitate overwintering of the virus. Herein, we report the first experimental infection of magpie (Pica pica) with two strains of West Nile virus, lineages 1 (NY-99) and 2 (SRB Novi-Sad/12), which are currently circulating in Europe. Magpies were highly susceptible to WNV infection, with similar low survival rates (30% and 42.8%) for both lineages. All infected magpies developed viremia detectable at 3 days post-infection with titers above those necessary for successful transmission of WNV to a mosquito. Neutralizing antibodies were detected at all time points analyzed (from 7 to 17 days post-infection). WNV genome was detected in the brains and hearts of all magpies that succumbed to the infection, and, in some of the surviving birds. WNV-RNA was amplified from swabs (oral and cloacal) at 3, 6 and 7 days post-infection and feather pulps, from 3 to 17 days post-infection, of infected animals. Even more, infectious virus was recovered from swabs up to 7 days post-infection and from feather pulps up to 10 days post infection. Sham-infected control animals were negative for viremia, viral RNA, and antibodies. These results suggest that the magpie, which is one of the most abundant corvid species in Europe, could represent a source of WNV transmission for birds and humans. Our observations shed light on the pathogenesis, transmission, and ecology of WNV and can benefit the implementation of surveillance and control programs.

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., Competing Interests: The authors declare no conflict of interest.

Background: We recently described the genotype/phenotype features of all Spanish patients diagnosed with McArdle disease as of January 2011 (n = 239, prevalence of ~1/167,000) (J Neurol Neurosurg Psychiatry 2012;83:322-8). Several caveats were however identified suggesting that the prevalence of the disease is actually higher., Methods: We have now updated main genotype/phenotype data, as well as potential associations within/between them, of all Spanish individuals currently diagnosed with McArdle disease (December 2016)., Results: Ninety-four new patients (all Caucasian) have been diagnosed, yielding a prevalence of ~1/139,543 individuals. Around 55% of the mutated alleles have the commonest PYGM pathogenic mutation p.R50X, whereas p.W798R and p.G205S account for 10 and 9% of the allelic variants, respectively. Seven new mutations were identified: p.H35R, p.R70C, p.R94Q, p.L132WfsX163, p.Q176P, p.R576Q, and c.244-3&#95;244-2CA. Almost all patients show exercise intolerance, the second wind phenomenon and high serum creatine kinase activity. There is, however, heterogeneity in clinical severity, with 8% of patients being asymptomatic during normal daily life, and 21% showing limitations during daily activities and fixed muscle weakness. A major remaining challenge is one of diagnosis, which is often delayed until the third decade of life in 72% of new patients despite the vast majority (86%) reporting symptoms before 20 years. An important development is the growing proportion of those reporting a 4-year improvement in disease severity (now 34%) and following an active lifestyle (50%). Physically active patients are more likely to report an improvement after a 4-year period in the clinical course of the disease than their inactive peers (odds ratio: 13.98; 95% confidence interval: 5.6, 34.9; p < 0.001). Peak oxygen uptake is also higher in the former (20.7 ± 6.0 vs. 16.8 ± 5.3 mL/kg/min, p = 0.0013). Finally, there is no association between PYGM genotype and phenotype manifestation of the disease., Conclusions: The reported prevalence of McArdle disease grows exponentially despite frequent, long delays in genetic diagnosis, suggesting that many patients remain undiagnosed. Until a genetic cure is available (which is not predicted in the near future), current epidemiologic data support that adoption of an active lifestyle is the best medicine for these patients.

Flaviviruses are RNA viruses that constitute a worrisome threat to global human and animal health. Zika virus (ZIKV), which was initially reported to cause a mild disease, recently spread in the Americas, infecting millions of people. During this recent epidemic, ZIKV infection has been linked to serious neurological diseases and birth defects, specifically Guillain-Barrè syndrome (GBS) and microcephaly. Because information about ZIKV immunity remains scarce, we assessed the humoral response of immunocompetent mice to infection with three viral strains of diverse geographical origin (Africa, Asia and America). No infected animals showed any sign of disease or died after infection. However, specific neutralizing antibodies were elicited in all infected mice. Considering the rapid expansion of ZIKV throughout the American continent and its co-circulation with other medically relevant flaviviruses, such as West Nile virus (WNV), the induction of protective immunity between ZIKV and WNV was analyzed. Remarkably, protection after challenge with WNV was observed in mice previously infected with ZIKV, as survival rates were significantly higher than in control mice. Moreover, previous ZIKV infection enhanced the humoral immune response against WNV. These findings may be relevant in geographical areas where both ZIKV and WNV co-circulate, as well as for the future development of broad-spectrum flavivirus vaccines.

Food allergy is a common disease affecting approximately 8% of children and 5% of adults. The prevalence has increased over the last two decades, suggesting an important environmental contribution to susceptibility. Studies have identified mode of birth, pet exposure, and having older siblings as being significant risk modifying factors in the development of food allergy. With the discovery that these factors significantly impact the composition of the intestinal microbiome, which is known to play a critical role in shaping the immune system, recent studies have begun to address the role of the intestinal microbiota in the development of food allergy. Studies in human cohorts support a dysbiosis in food allergy, and limited data suggest that this dysbiosis occurs early in life, preceding the onset of sensitization. Studies from animal models have clearly shown that the composition of the intestinal microbiota confers susceptibility to food allergy, and that there are organisms such as Clostridia species that are protective in the development of food allergy. Our understanding of microbial regulation of food allergy is in its nascency, but the state of the field supports an important contribution of intestinal microbes to susceptibility. Challenges going forward are to identify commensal-derived microorganisms that could be used therapeutically to prevent or perhaps treat food allergy., Competing Interests: The authors have read the journal’s policy on conflict of interest disclosure, and have none to declare. The authors have read the journal’s authorship agreement and the manuscript has been reviewed and approved by all named authors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) is a flavivirus ( Flaviviridae family) transmitted mainly by Aedes mosquitoes. The virus was restricted to the African continent until its spread to south-east Asia in the 1980's, the Micronesia in 2007, the French Polynesia in 2013 and, more recently in the Americas in 2015, where, up to date, the World Health Organization (WHO) has estimated about 3-4 million total cases of ZIKV infection. During outbreaks in the French Polynesia and Brazil in 2013 and 2015, respectively, national health authorities reported potential neurological complications of ZIKV disease, chiefly an upsurge in Guillain-Barré syndrome, which coincided with ZIKV outbreaks. On the other hand, the emergence of ZIKV in Brazil has been associated with a striking increase in the number of reported cases of microcephaly in fetus and newborns, twenty times higher than in that reported in previous years. While investigations are currently assessing whether there is an actual association between neurological complications and ZIKV infections, the evidence was enough worrisome for WHO to declare a public health emergency of international concern. Here we present an updated review addressing what is currently known about the possible association between ZIKV infection and the development of severe neurological disorders., Competing Interests: Conflict-of-interest statement: The authors report no conflict of interest.

After the introduction of West Nile virus (WNV) into North America, bird mortalities associated with West Nile disease have dramatically increased in this continent and, to a lesser extent, in Europe. The different West Nile disease incidence in birds in these 2 continents demands an explanation, and experimental studies can provide important information. The authors inoculated thirteen 9-week-old red-legged partridges (Alectoris rufa) with 10(7)plaque-forming units of a WNV strain isolated in New York in 1999. The objective was to study the pathogenesis of the infection in a native Euro-Mediterranean bird species with a WNV strain known to be highly pathogenic for numerous native American bird species. Additionally, the authors evaluated the dynamics of inflammatory cell activation and recruitment into the brain. WNV was detected in tissues 3 days postinoculation (dpi), and the birds developed macroscopic and microscopic lesions. Two partridges succumbed to the disease. The most affected tissues were the heart, brain, and spinal cord. The main microscopic findings were the presence of mononuclear infiltrates in the heart and brain, gliosis, and degeneration and necrosis of cardiomyocytes and neurons. These lesions were aggravated in the birds that died or were euthanized 7 dpi or later. In the brain, there was an upregulation of microglial cells and astrocytes and an increase in the number of T cells, especially after 7 dpi. These results show that this WNV strain is of moderate virulence for the red-legged partridge and that WNV-infected red-legged partridges develop an immune cell response in the brain similar to that of mammals., (© The Author(s) 2015.)

Food allergy has rapidly increased in prevalence, suggesting an important role for environmental factors in disease susceptibility. The immune response of food allergy is characterized by IgE production, and new findings from mouse and human studies indicate an important role of the cytokine IL-9, which is derived from both T cells and mast cells, in disease manifestations. Emerging evidence suggests that route of exposure to food, particularly peanut, is important. Exposure through the skin promotes sensitization while early exposure through the gastrointestinal tract promotes tolerance. Evidence from mouse studies indicate a role of the microbiome in development of food allergy, which is supported by correlative human studies showing a dysbiosis in food allergy. There is no approved treatment for food allergy, but emerging therapies are focused on allergen immunotherapy to provide desensitization, while pre-clinical studies are focused on using adjuvants or novel delivery approaches to improve efficacy and safety of immunotherapy., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Usutu virus (USUV) is a mosquito-borne flavivirus whose circulation had been confined to Africa since it was first detected in 1959. However, in the last decade USUV has emerged in Europe causing episodes of avian mortality and sporadic severe neuroinvasive infections in humans. Remarkably, adult laboratory mice exhibit limited susceptibility to USUV infection, which has impaired the analysis of the immune responses, thus complicating the evaluation of virus-host interactions and of vaccine candidates against this pathogen. In this work, we showed that mice deficient in the alpha/beta interferon receptor (IFNAR (-/-) mice) were highly susceptible to USUV infection and provided a lethal challenge model for vaccine testing. To validate this infection model, a plasmid DNA vaccine candidate encoding the precursor of membrane (prM) and envelope (E) proteins of USUV was engineered. Transfection of cultured cells with this plasmid resulted in expression of USUV antigens and the assembly and secretion of small virus-like particles also known as recombinant subviral particles (RSPs). A single intramuscular immunization with this plasmid was sufficient to elicit a significant level of protection against challenge with USUV in IFNAR (-/-) mice. The characterization of the humoral response induced revealed that DNA vaccination primed anti-USUV antibodies, including neutralizing antibodies. Overall, these results probe the suitability of IFNAR (-/-) mice as an amenable small animal model for the study of USUV host virus interactions and vaccine testing, as well as the feasibility of DNA-based vaccine strategies for the control of this pathogen., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Since the beginning of this century, humanity has been facing a new emerging, or re-emerging, virus threat almost every year: West Nile, Influenza A, avian flu, dengue, Chikungunya, SARS, MERS, Ebola, and now Zika, the latest newcomer. Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes, was identified in 1947 in a sentinel monkey in Uganda, and later on in humans in Nigeria. The virus was mainly confined to the African continent until it was detected in south-east Asia the 1980's, then in the Micronesia in 2007 and, more recently in the Americas in 2014, where it has displayed an explosive spread, as advised by the World Health Organization, which resulted in the infection of hundreds of thousands of people. ZIKV infection was characterized by causing a mild disease presented with fever, headache, rash, arthralgia, and conjunctivitis, with exceptional reports of an association with Guillain-Barre syndrome (GBS) and microcephaly. However, since the end of 2015, an increase in the number of GBS associated cases and an astonishing number of microcephaly in fetus and new-borns in Brazil have been related to ZIKV infection, raising serious worldwide public health concerns. Clarifying such worrisome relationships is, thus, a current unavoidable goal. Here, we extensively review what is currently known about ZIKV, from molecular biology, transmission routes, ecology, and epidemiology, to clinical manifestations, pathogenesis, diagnosis, prophylaxis, and public health.

West Nile virus (WNV) is a mosquito-borne flavivirus maintained in a transmission cycle between mosquitoes and birds, but it can also infect other vertebrates, including humans, in which it can cause neuroinvasive diseases. To date, no licensed vaccine or therapy for human use against this pathogen is yet available. A recent approach to search for new antiviral agent candidates is the assessment of long-used drugs commonly administered by clinicians to treat human disorders in drug antiviral development. In this regard, as patients with West Nile encephalitis frequently develop symptoms and features of parkinsonism, and cellular factors altered in parkinsonism, such as alpha-synuclein, have been shown to play a role on WNV infection, we have assessed the effect of four drugs (L-dopa, Selegiline, Isatin, and Amantadine), that are used as therapy for Parkinson's disease in the inhibition of WNV multiplication. L-dopa, Isatin, and Amantadine treatments significantly reduced the production of infectious virus in all cell types tested, but only Amantadine reduced viral RNA levels. These results point to antiparkinsonian drugs as possible therapeutic candidates for the development of antiviral strategies against WNV infection.

Flaviviruses are RNA viruses that constitute a worrisome threat to global human and animal health. In Europe, West Nile virus (WNV) outbreaks have dramatically increased in number and severity in recent years, with dozens of human and horse deaths and a high avian mortality across the continent. Besides WNV, the only clinically relevant mosquito-borne flavivirus detected so far in Europe has been the Usutu virus (USUV), which after being reported for the first time in Austria in 2001, quickly spread across Europe, causing a considerable number of bird deaths and neurological disorders in a few immunocompromised patients. Even though USUV infects multiple avian species that develop antibodies, there is little information about USUV susceptibility, pathogenicity and cross-reactive immunity. Here, the susceptibility of suckling and adult mice to USUV infection and the induction of cross-protective immunity against WNV challenge have been addressed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, but it also infects many other vertebrates, including humans and horses, in which it can induce severe neurological diseases; however, data about virus circulation in other mammals is scarce. WNV has a history of recent outbreaks in Europe, including Serbia, where it was identified for the first time in 2010 in mosquitoes and in 2012 in birds and humans, being responsible for over 300 confirmed human cases and 35 deaths there along 2013. To assess WNV circulation among mammals in the country, 688 samples obtained from 279 farm pigs, 318 wild boars, and 91 roe deer were investigated for the presence of antibodies to WNV by enzyme-linked immunosorbent assay (ELISA) and viral neutralization test (VNT), and the specificity of their reactivity was assayed against Usutu virus (USUV). ELISA-reactive sera were identified in 43 (15.4%) pigs, 56 (17.6%) wild boars, and 17 (18.7%) roe deer. Of these, 6 (14%), 33 (59%), and 4 (23.5%) respectively, neutralized WNV. One out of the 45 ELISA negative sera tested, from a roe deer, neutralized WNV. Cross-reactivity neutralization test indicated that all deer and pigs neutralizing sera were WNV specific, while in 5 (15.2%) of the wild boar samples the specificity could not be established. Four wild boar sera showed USUV specificity. All these data confirm the circulation of both flaviviruses in Serbia, and highlight the need for the implementation of global coordinated surveillance programs in the region., (Copyright © 2015 Elsevier B.V. All rights reserved.)

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for outbreaks of meningitis and encephalitis. Whereas the activation of autophagy in cells infected with other flaviviruses is well known, the interaction of WNV with the autophagic pathway still remains unclear and there are reports describing opposite findings obtained even analyzing the same viral strain. To clarify this controversy, we first analyzed the induction of autophagic features in cells infected with a panel of WNV strains. WNV was determined to induce autophagy in a strain dependent manner. We observed that all WNV strains or isolates analyzed, except for the WNV NY99 used, upregulated the autophagic pathway in infected cells. Interestingly, a variant derived from this WNV NY99 isolated from a persistently infected mouse increased LC3 modification and aggregation. Genome sequencing of this variant revealed only two non-synonymous nucleotide substitutions when compared to parental NY99 strain. These nucleotide substitutions introduced one amino acid replacement in NS4A and other in NS4B. Using genetically engineered viruses we showed that introduction of only one of these replacements was sufficient to upregulate the autophagic pathway. Thus, in this work we have shown that naturally occurring point mutations in the viral non-structural proteins NS4A and NS4B confer WNV with the ability to induce the hallmarks of autophagy such as LC3 modification and aggregation. Even more, the differences on the induction of an autophagic response observed among WNV variants in infected cells did not correlate with alterations on the activation of the unfolded protein response (UPR), suggesting an uncoupling of UPR and autophagy during flavivirus infection. The findings here reported could help to improve the knowledge of the cellular processes involved on flavivirus-host cell interactions and contribute to the design of effective strategies to combat these pathogens.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.

Using eye samples of nine 9-week-old experimentally West Nile virus (WNV)-infected red-legged partridges (Alectoris rufa), time course of lesions and WNV antigen appearance in ocular structures were examined. In addition, eye samples of 6 red-legged partridges and 3 common pheasants (Phasianus colchicus) naturally infected with Bagaza virus (BAGV) were used to study lesions and flavivirus antigen distribution in relation to apparent blindness in the former. The rapid onset of microscopic lesions and early presence of viral antigen in the eye of experimentally WNV-infected partridges, prior to the central nervous system involvement, suggested hematogenous spread of the virus into the eye. BAGV-infected partridges had a more pronunced inflammatory reaction and more widespread flavivirus antigen distribution in the retina compared with pheasants and experimentally fatally WNV-infected partridges. Our results suggest that flavivirus replication and development of lesions in ocular structures of gallinaceous game birds vary with the specific virus and host species involved., (© The Author(s) 2014.)

Introduction: Hepatitis E virus (HEV) infection is rarely reported in industrialized countries, but recent studies have revealed quite variable seroprevalence rates among European populations, including blood donors. In Serbia, very limited data about HEV seroprevalence are available. This study aimed to determine the prevalence of anti-HEV IgG antibodies and HEV RNA in the sera of volunteer blood donors in Serbia., Methodology: Serum samples from 200 volunteer blood donors were tested for the presence of anti-HEV IgG by enzyme-linked immunosorbent assay (ELISA) using ORF-2 HEV genotype 3 recombinant proteins as antigen, and for the presence of HEV RNA by nested reverse transcriptase polymerase chain reaction (RT-PCR)., Results: In total, 15% of the volunteer blood donors were seropositive. The prevalence increased with age; 21.5%, 14.2%, and 5.4% HEV seroprevalence rates were found in individuals older than 51 years, between 31 and 50 years, and in those younger than 30 years of age, respectively. However, no HEV RNA was detected in any of the individuals analyzed., Conclusions: The prevalence of anti-HEV IgG among blood donors as representatives of the general population is quite high in Serbia compared to data from many European countries. One of the reasons for this could be the high prevalence of HEV among Serbian pigs and the traditional consumption of piglet meat in the country. The relatively high HEV seroprevalence found among Serbian blood donors indicates the need for further investigation.

West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV. Importance: West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed for human use. Since the multiplication of this virus is associated with rearrangements of host cell membranes, we analyzed the effect of WNV infection on different cellular lipids that constitute important membrane components. The levels of multiple lipid species were increased in infected cells, pointing to the induction of major alterations of cellular lipid metabolism by WNV infection. Interestingly, certain sphingolipids, which were increased in infected cells, were also enriched in the lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of sphingolipids in the production of WNV by means of functional analyses. This study provides new insight into the formation of flavivirus infectious particles and the involvement of sphingolipids in the WNV life cycle., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus. High WNV virulence was mainly associated with lineage 1 strains, but recent outbreaks have unveiled circulation of highly virulent lineage 2 strains. Co-expression of flavivirus prM and E glycoproteins drives the assembly of recombinant subviral particles (RSPs) that share antigenic features with virions. Mouse immunization with lineage 1 WNV RSPs induced a potent humoral response against WNV with production of neutralizing antibodies. A single inoculation of RSPs formulated with Al(OH)3 as adjuvant protected mice against a lethal challenge with WNV strains from lineage 1 or 2. The cross-reactivity of the response elicited by these RSPs was analyzed against the related flavivirus Usutu virus (USUV), which shares multiple ecological and antigenic features with WNV. Immunization with WNV-RSPs increased specific, although low, antibody titers found upon subsequent USUV infection.

The Flavivirus is a genus of RNA viruses that includes multiple long known human, animal, and zoonotic pathogens such as Dengue virus, yellow fever virus, West Nile virus, or Japanese encephalitis virus, as well as other less known viruses that represent potential threats for human and animal health such as Usutu or Zika viruses. Flavivirus replication is based on endoplasmic reticulum-derived structures. Membrane remodeling and accumulation of viral factors induce endoplasmic reticulum stress that results in activation of a cellular signaling response termed unfolded protein response (UPR), which can be modulated by the viruses for their own benefit. Concomitant with the activation of the UPR, an upregulation of the autophagic pathway in cells infected with different flaviviruses has also been described. This review addresses the current knowledge of the relationship between endoplasmic reticulum stress, UPR, and autophagy in flavivirus-infected cells and the growing evidences for an involvement of these cellular pathways in the replication and pathogenesis of these viruses.

Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection.

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n=28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n=29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Food allergy is an increasing problem in western countries, with strict avoidance being the only available reliable treatment. However, accidental ingestion can occur and anaphylactic reactions still happen. In recent years, many efforts have been made to better understand the humoral and cellular mechanisms involved in food allergy, and to improve the strategies for diagnosis and treatment. This review focuses on IgE-mediated food hypersensitivity and provides an overview of the diagnostic strategies and treatment advances. Specific immunotherapy, including different routes of administration and allergen sources, such as natural, recombinant and T-cell epitopes, are analyzed in detail. Other treatments such as anti-IgE monoclonal antibody therapy, adjuvant therapy and Chinese herbs will also be described.