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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.

Abstract 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.

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.

ABSTRACTFlaviviruses 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 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.

The mosquito-borne West Nile virus (WNV) is a highly neurovirulent Flavivirus currently representing an emergent zoonotic concern. WNV cycles in nature between mosquito vectors and birds that act as amplifier hosts and play an essential role in virus ecology, being, thus, WNV a threat to many species. Availability of an efficient avian vaccine would benefit certain avian populations, both birds grown for hunting and restocking activities, as well as endangered species in captive breeding projects, wildlife reservations, and recreation installations, and would be useful to prevent and contain outbreaks. Avian vaccination would be also of interest to limit WNV spillover to humans or horses from susceptible bird species that live in urbanized landscapes, like magpies. Herein, we have addressed the efficacy of a single dose of a WNV recombinant subviral particle (RSP) vaccine in susceptible magpie (Pica pica). The protective capacity of the RSP-based vaccine was demonstrated upon challenge of magpies with 5 × 103 plaque forming units of a neurovirulent WNV strain. A significant improvement in survival rates of immunized birds was recorded when compared to vehicle-inoculated animals (71.4 vs. 22.2%, respectively). Viremia, which is directly related to the capacity of a host to be competent for virus transmission, was reduced in vaccinated animals, as was the presence of infectious virus in feather follicles. Bird-to-bird transmission was recorded in three of six unchallenged (contact) magpies housed with non-vaccinated WNV-infected birds, but not in contact animals housed with vaccinated WNV-infected magpies. These results demonstrate the protective efficacy of the RSP-based vaccine in susceptible birds against WNV infection and its value in controlling the spread of the virus.

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.

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.Emerging Microbes & Infections (2017) 6, e81; doi:10.1038/emi.2017.68; published online 20 September 2017

In the frame of a Flemish wildlife surveillance in 2013, a serological screening was performed on sera from wild boar (Sus scrofa; n=238) in order to detect tick-borne encephalitis virus (TBEV)-specific antibodies. Neutralising antibodies were titrated with a seroneutralisation test (SNT), using two cut-off titres (1/10–1/15). Seven wild boars were found TBEV-seropositive and showed moderate (>1/15) to high (>1/125) SNT-titres; three individuals had borderline results (1/10–1/15). This study demonstrated the presence of TBEV-specific antibodies in wild boar and highlighted potential TBEV-foci in Flanders. Additional surveillance including direct virus testing is now recommended.

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.

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.

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.

El objetivo de este estudio fue comparar el comportamiento de El objetivo de este estudio fue comparar el comportamiento de El objetivo de este estudio fue comparar el comportamiento de IFN-ɤ e IL-4 en ratones inoculados con proteína G recombinante del virus de la rabia, que se expresa en maíz transgénico o baculovirus. Para este propósito, grupos de ratones se inocularon de la siguiente manera: Grupo 1, vacuna contra el virus de la rabia inactivado vía im; Grupo 2, proteína G recombinante derivada de plantas por vía oral; Grupo 3, proteína G expresada en baculovirus vía im; Grupo 4, proteína G expresada en baculovirus por vía oral; Grupo 5, maíz no transformado por vía oral. Los niveles de IFN-ɤ e IL-4 y los anticuerpos específicos se evaluaron cada 15 días. El desafío se realizó a los 60 días post inoculación (pi). Los grupos 1 y 3 promovieron una mejor respuesta humoral. Por otro lado, los resultados mostraron que los mismos grupos mostraron los mejores niveles de IFN-ɤ en el día 10 pi; mientras que la IL-4 se detectó en el día 15 pi. Para el estudio de supervivencia, el 83 % de los ratones inmunizados con vacuna inactivada, maíz y los inoculados im con extractos de baculovirus sobrevivieron la infección viral. La proteína G de la rabia recombinante expresada en baculovirus promovió IFN-ɤ y los anticuerpos de una manera similar a la vacuna de rabia inactivada. A pesar de esto los ratones alimentados con maíz transgénico sobrevivieron al desafío en el mismo porcentaje que el grupo 3. e IL-4 en ratones inoculados con proteína G recombinante del virus de la rabia, que se expresa en maíz transgénico o baculovirus. Para este propósito, grupos de ratones se inocularon de la siguiente manera: Grupo 1, vacuna contra el virus de la rabia inactivado vía im; Grupo 2, proteína G recombinante derivada de plantas por vía oral; Grupo 3, proteína G expresada en baculovirus vía im; Grupo 4, proteína G expresada en baculovirus por vía oral; Grupo 5, maíz no transformado por vía oral. Los niveles de IFN-ɤ e IL-4 y los anticuerpos específicos se evaluaron cada 15 días. El desafío se realizó a los 60 días post inoculación (pi). Los grupos 1 y 3 promovieron una mejor respuesta humoral. Por otro lado, los resultados mostraron que los mismos grupos mostraron los mejores niveles de IFN-ɤ en el día 10 pi; mientras que la IL-4 se detectó en el día 15 pi. Para el estudio de supervivencia, el 83 % de los ratones inmunizados con vacuna inactivada, maíz y los inoculados im con extractos de baculovirus sobrevivieron la infección viral. La proteína G de la rabia recombinante expresada en baculovirus promovió IFN-ɤ y los anticuerpos de una manera similar a la vacuna de rabia inactivada. A pesar de esto los ratones alimentados con maíz transgénico sobrevivieron al desafío en el mismo porcentaje que el grupo 3. e IL-4 en ratones inoculados con proteína G recombinante del virus de la rabia, que se expresa en maíz transgénico o baculovirus. Para este propósito, grupos de ratones se inocularon de la siguiente manera: Grupo 1, vacuna contra el virus de la rabia inactivado vía im; Grupo 2, proteína G recombinante derivada de plantas por vía oral; Grupo 3, proteína G expresada en baculovirus vía im; Grupo 4, proteína G expresada en baculovirus por vía oral; Grupo 5, maíz no transformado por vía oral. Los niveles de IFN-ɤ e IL-4 y los anticuerpos específicos se evaluaron cada 15 días. El desafío se realizó a los 60 días post inoculación (pi). Los grupos 1 y 3 promovieron una mejor respuesta humoral. Por otro lado, los resultados mostraron que los mismos grupos mostraron los mejores niveles de IFN-ɤ en el día 10 pi; mientras que la IL-4 se detectó en el día 15 pi. Para el estudio de supervivencia, el 83 % de los ratones inmunizados con vacuna inactivada, maíz y los inoculados im con extractos de baculovirus sobrevivieron la infección viral. La proteína G de la rabia recombinante expresada en baculovirus promovió IFN-ɤ y los anticuerpos de una manera similar a la vacuna de rabia inactivada. A pesar de esto los ratones alimentados con maíz transgénico sobrevivieron al desafío en el mismo porcentaje que el grupo 3.