Miguel A. Martín-Acebes

West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.

West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. As in the case of other plus-stranded RNA viruses, WNV replication is associated to intracellular membrane rearrangements. Based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. As these processes are related to lipid metabolism, fatty acid synthesis, as well as generation of a specific lipid microenvironment enriched in phosphatidylinositol-4-phosphate (PI4P), has been associated to it in other viral models. In this study, intracellular membrane rearrangements following infection with a highly neurovirulent strain of WNV were addressed by means of electron and confocal microscopy. Infection of WNV, and specifically viral RNA replication, were dependent on fatty acid synthesis, as revealed by the inhibitory effect of cerulenin and C75, two pharmacological inhibitors of fatty acid synthase, a key enzyme of this process. However, WNV infection did not induce redistribution of PI4P lipids, and PI4P did not localize at viral replication complex. Even more, WNV multiplication was not inhibited by the use of the phosphatidylinositol-4-kinase inhibitor PIK93, while infection by the enterovirus Coxsackievirus B5 was reduced. Similar features were found when infection by other flavivirus, the Usutu virus (USUV), was analyzed. These features of WNV replication could help to design specific antiviral approaches against WNV and other related flaviviruses.

Zika Virus: the Latest Newcomer

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.

Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy

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.

West Nile virus: A re-emerging pathogen revisited

West Nile virus (WNV), a flavivirus of the Flaviviridae family, is maintained in nature in an enzootic transmission cycle between avian hosts and ornithophilic mosquito vectors, although the virus occasionally infects other vertebrates. WNV causes sporadic disease outbreaks in horses and humans, which may result in febrile illness, meningitis, encephalitis and flaccid paralysis. Until recently, its medical and veterinary health concern was relatively low; however, the number, frequency and severity of outbreaks with neurological consequences in humans and horses have lately increased in Europe and the Mediterranean basin. Since its introduction in the Americas, the virus spread across the continent with worrisome consequences in bird mortality and a considerable number of outbreaks among humans and horses, which have resulted in the largest epidemics of neuroinvasive WNV disease ever documented. Surprisingly, its incidence in human and animal health is very different in Central and South America, and the reasons for it are not yet understood. Even though great advances have been obtained lately regarding WNV infection, and although efficient equine vaccines are available, no specific treatments or vaccines for human use are on the market. This review updates the most recent investigations in different aspects of WNV life cycle: molecular virology, transmission dynamics, host range, clinical presentations, epidemiology, ecology, diagnosis, control, and prevention, and highlights some aspects that certainly require further research.

The Composition of West Nile Virus Lipid Envelope Unveils a Role of Sphingolipid Metabolism in Flavivirus Biogenesis

ABSTRACT 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.

First Serological Evidence of West Nile Virus Activity in Horses in Serbia

West Nile virus (WNV), the most widely distributed flavivirus worldwide, has lately reemerged in Europe, causing worrisome outbreaks in humans and horses. Serological analysis by enzyme-linked immunoassay and plaque reduction neutralization test showed for the first time in Serbia that 12% of 349 horses presented specific neutralizing WNV antibodies, which in one case also cross-neutralized Usutu virus (USUV). This is the first time that anti-USUV high neutralizing antibody titers are reported in horses. All these data indicate that WNV and USUV are circulating in the region and advise on the convenience of implementing surveillance programs.

A West Nile virus mutant with increased resistance to acid-induced inactivation

West Nile virus (WNV) is a mosquito-borne flavivirus responsible for epidemics of febrile illness, meningitis, encephalitis and flaccid paralysis. WNV gains entry into host cells through endocytosis. The acid pH inside endosomes triggers rapid conformational rearrangements of the flavivirus envelope (E) glycoprotein that result in fusion of the endosomal membrane with the virion envelope. Conformational rearrangements of the E glycoprotein can be induced by acid exposure in solution in the absence of target membranes, thus causing a loss of infectivity. Following a genetic approach to study this process, a WNV mutant with increased resistance to acid-induced inactivation was isolated and its complete genome was sequenced. A single amino acid substitution, T70I, in the E glycoprotein was found to be responsible for the increased acid resistance, which was linked to an increase in the sensitivity of infection to the chemical rise of endosomal pH, suggesting that the mutant required a more acid pH inside the endosomes for fusion. No alterations in viral infection kinetics, plaque size or induced mortality rates in mice of the mutant were noted. However, by means of virus competition assays, a reduction in viral fitness under standard culture conditions was observed for the mutant. These results provide new evidence of the adaptive flexibility to environmental factors--pH variation in this case--of WNV populations. Implications of the T70I replacement on the E glycoprotein structure-function relationship are discussed.

A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Lability and Confer Resistance to Acid-Dependent Uncoating Inhibition

ABSTRACT The acid-dependent disassembly of foot-and-mouth disease virus (FMDV) is required for viral RNA release from endosomes to initiate replication. Although the FMDV capsid disassembles at acid pH, mutants escaping inhibition by NH4Cl of endosomal acidification were found to constitute about 10% of the viruses recovered from BHK-21 cells infected with FMDV C-S8c1. For three of these mutants, the degree of NH4Cl resistance correlated with the sensitivity of the virion to acid-induced inactivation of its infectivity. Capsid sequencing revealed the presence in each of these mutants of a different amino acid substitution (VP3 A123T, VP3 A118V, and VP2 D106G) that affected a highly conserved residue among FMDVs located close to the capsid interpentameric interfaces. These residues may be involved in the modulation of the acid-induced dissociation of the FMDV capsid. The substitution VP3 A118V present in mutant c2 was sufficient to confer full resistance to NH4Cl and concanamycin A (a V-ATPase inhibitor that blocks endosomal acidification) as well as to increase the acid sensitivity of the virion to an extent similar to that exhibited by mutant c2 relative to the sensitivity of the parental virus C-S8c1. In addition, the increased propensity to dissociation into pentameric subunits of virions bearing substitution VP3 A118V indicates that this replacement also facilitates the dissociation of the FMDV capsid.

A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Resistance

ABSTRACT 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 (NH4Cl 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.

The Race To Find Antivirals for Zika Virus

ABSTRACT Zika virus (ZIKV), a flavivirus transmitted by mosquitoes, was an almost neglected pathogen until its introduction in the Americas in 2015 and its subsequent explosive spread throughout the continent, where it has infected millions of people. The virus has caused social and sanitary alarm, mainly due to its association with severe neurological disorders (Guillain-Barré syndrome and microcephaly in fetuses and newborns). Nowadays, no specific antiviral therapy against ZIKV is available. However, during the past months, a great effort has been made to search for antiviral candidates using different approaches and methodologies, ranging from testing specific compounds with known antiviral activity to the screening of libraries with hundreds of bioactive molecules. The identified antiviral candidates include drugs targeting viral components as well as cellular ones. Here, an updated review of what has been done in this line is presented.