A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation.

West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains.IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.

Synthesis of 4'-Substituted-2'-Deoxy-2'-α-Fluoro Nucleoside Analogs as Potential Antiviral Agents.

Nucleoside analogs are widely used for the treatment of viral diseases (Hepatitis B/C, herpes and human immunodeficiency virus, HIV) and various malignancies. ALS-8176, a prodrug of the 4'-chloromethyl-2'-deoxy-2'-fluoro nucleoside ALS-8112, was evaluated in hospitalized infants for the treatment of respiratory syncytial virus (RSV), but was abandoned for unclear reasons. Based on the structure of ALS-8112, a series of novel 4'-modified-2'-deoxy-2'-fluoro nucleosides were synthesized. Newly prepared compounds were evaluated against RSV, but also against a panel of RNA viruses, including Dengue, West Nile, Chikungunya, and Zika viruses. Unfortunately, none of the compounds showed marked antiviral activity against these viruses.

West Nile Virus fidelity modulates the capacity for host cycling and adaptation.

The fidelity of flaviviruses is thought to be tightly regulated for optimal fitness within and between hosts. West Nile virus (WNV) high-fidelity (HiFi) mutations V793I and G806R within the RNA-dependent RNA polymerase, and low-fidelity (LoFi) mutation T248I within the methyltransferase, were previously shown to attenuate infectivity and replicative fitness in Culex mosquitoes and Culex tarsalis (CXT) cells but not in mammalian cells. We hypothesized that fidelity alterations would modify adaptation and maintenance in a host-specific manner. To test this hypothesis, wild-type (WT), HiFi (V793I/G806R) and LoFi (T248I) variants were sequentially passaged eight times in avian (PDE) or mosquito cells, or alternately between the two. Initial characterization confirmed that fidelity mutants are attenuated in mosquito, but not avian, cells. Deep sequencing revealed mutations unique to both cell lines and fidelity mutants, including ENV G1378A, a mutation associated with avian cell adaptation. To characterize maintenance and adaptation, viral outputs were monitored throughout passaging and viral fitness was assessed. The results indicate that fidelity mutants can at times recover fitness during mosquito cell passage, but remain attenuated relative to WT. Despite similar initial fitness, LoFi mutants were impaired during sequential passage in avian cells. Conversely, HiFi mutants passaged in avian cells showed increased adaptation, suggesting that increased fidelity may be advantageous in avian hosts. Although some adaptation occurred with individual mutants, the output titres of fidelity mutants were on average lower and were often lost during host switching. These data confirm that arbovirus fidelity is likely fine-tuned to maximize survival in disparate hosts.

El virus de nilo occidental y sus vectores: reseña histórica, biología y ecología de aedes albopictus, culex pipiens y cx. quinquefasciatus / Western Nile virus and its vectors: a historic review, biology and ecology of Aedes albopictus, Culex pipiens and Cx. Quinquefasciatus

Resumen Este trabajo obedece a la primera iniciativa para compilar el conocimiento de los mosquitos vectores del Virus del Nilo Occidental en Costa Rica, como parte del primer Congreso para el estudio de la epidemiología de este arbovirus. Se incluye la revisión de las bases de datos de mayor prestigio y mayor cantidad de artículos para completar la información referente a lo que se ha informado acerca de las especies de mosquitos en Costa Rica, acerca de los cuales la información es fragmentaria y de difícil acceso en muchos casos. Se recomienda incentivar la investigación en este importante grupo de insectos, además de la necesidad de estudios de la ecología de estas especies en nuestro país. Se incluye una clave para los principales géneros de mosquitos hallados en Costa Rica en los cuales hay especies informadas como posibles vectores en otras latitudes. Se hace una sinopsis de la necesidad de incluir factores psicosociales en el diseño de las campañas educativas.

Abstract This work is the first initiative to compile the knowledge of mosquito vectors of West Nile Virus in Costa Rica, as part of the first Congress to study the epidemiology of this arbovirus. It includes a review of the most prestigious databases and a greater number of articles to complete the information regarding what has been reported about mosquito species in Costa Rica, about which the information is fragmentary and difficult to access in many cases. It is recommended to encourage research in this important group of insects, in addition to the need for studies of the ecology of these species in our country. A key is included for the main genera of mosquitoes found in Costa Rica in which there are species reported as possible vectors in other latitudes. A synopsis is made of the need to include psychosocial factors in the design of educational campaigns.

Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance.

CD8+ T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8+ T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a-/-) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8+ T cells isolated from Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 postinfection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8+ T cell cytotoxicity, which may have broad implications in other microbial infections and cancers. IMPORTANCE: Interleukin-17A (IL-17A) and CD8+ T cells regulate diverse immune functions in microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while CD8+ T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8+ T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8+ T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces the viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8+ T cell axis may also have broad implications for immunity to other microbial infections and cancers, where CD8+ T cell functions are crucial.

Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments.

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC50] = 0.03 µM) and Semliki Forest virus (SFV; EC50 = 0.11 µM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate.

Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control.

The fight against mosquito-borne diseases is a challenge of huge public health importance. To our mind, 2015 was an extraordinary year for malaria control, due to three hot news: the Nobel Prize to Youyou Tu for the discovery of artemisinin, the development of the first vaccine against Plasmodium falciparum malaria [i.e. RTS,S/AS01 (RTS,S)], and the fall of malaria infection rates worldwide, with special reference to sub-Saharan Africa. However, there are major challenges that still deserve attention, in order to boost malaria prevention and control. Indeed, parasite strains resistant to artemisinin have been detected, and RTS,S vaccine does not offer protection against Plasmodium vivax malaria, which predominates in many countries outside of Africa. Furthermore, the recent outbreaks of Zika virus infections, occurring in South America, Central America and the Caribbean, represent the most recent of four arrivals of important arboviruses in the Western Hemisphere, over the last 20 years. Zika virus follows dengue (which slyly arrived in the hemisphere over decades and became more aggressive in the 1990s), West Nile virus (emerged in 1999) and chikungunya (emerged in 2013). Notably, there are no specific treatments for these arboviruses. The emerging scenario highlights that the effective and eco-friendly control of mosquito vectors, with special reference to highly invasive species such as Aedes aegypti and Aedes albopictus, is crucial. The concrete potential of screening plant species as sources of metabolites for parasitological purposes is worthy of attention, as elucidated by the Y. Tu's example. Notably, plant-borne molecules are often effective at few parts per million against Aedes, Ochlerotatus, Anopheles and Culex young instars, can be used for the rapid synthesis of mosquitocidal nanoformulations and even employed to prepare cheap repellents with low human toxicity. In addition, behaviour-based control tools relying to the employ of sound traps and the manipulation of swarming behaviour (i.e. "lure and kill" approach) are discussed. The importance of further research on the chemical cues routing mosquito swarming and mating dynamics is highlighted. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in the presence of ultra-low quantities of nanoformulated botanicals, which boost their predation rates.

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

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

[L-Lysine-α-Oxidase in vitro Activity in Experiments on Models of Viruses Sindbis, Forest-Spring Encephalitis, Western Nile, Tyaginya and Dhori].

The antitumor effect of L-lysine-α-oxidase from the culture fluid of Trichoderma harzianum Rifai F-180 was investigated for the first time. The in vitro studies revealed its high activity on a model of the forest-spring encephalitis virus and no activity against the Sindbis, Western Nile, Tyaginya and Dhori viruses.

Combined treatment of adenosine nucleoside inhibitor NITD008 and histone deacetylase inhibitor vorinostat represents an immunotherapy strategy to ameliorate West Nile virus infection.

West Nile virus (WNV), a member of the Flaviviridae family, is the leading cause of viral encephalitis in the United States. Despite efforts to control the spread of WNV, there has been an increase in the number of outbreaks and clinical cases with neurological problems. There are no antiviral compounds currently in trials for WNV. NITD008 is an adenosine analogue inhibitor that interrupts the RNA-dependent RNA polymerase of flaviviruses. Previous studies demonstrated NITD008 as a potent antiviral for dengue virus, however this drug was associated with preclinical toxicity. The ability of NITD008 to block WNV replication is only shown in Vero cells. Neuroinflammation is also a major cause of the WNV-associated pathology, therefore we evaluated the effect of NITD008 and a newly characterized anti-inflammatory drug vorinostat (SAHA), a histone deacetylase inhibitor, on WNV replication and disease progression in a mouse model. When administered at 10 and 25mg/kg at days 1-6 after WNV infection in C57BL/6 mice, NITD008 conferred complete protection from clinical symptoms and death, which correlated with reduced viral load in the serum and restriction of virus-CNS entry. Delay of NITD008 treatment to days 3-6 and days 5-9 after infection, when WNV replication was high in the periphery and brain, resulted in the gradual loss of protection against WNV infection. However, co-treatment with SAHA and NITD008 during the CNS phase of disease improved disease outcome significantly by reducing inflammation and neuronal death. Our results support potential synergistic effect of combination therapy of NITD008 with SAHA for the treatment of WNV encephalitis.

[Antiviral activity in vitro and pharmacokinetics of HCV entry inhibitor AVR560].

Several novel compounds were found to be potent inhibitors of the HCV (JFH-1 isolate) infection in vitro. Human serum did not significantly reduce antiviral activity of the lead compound, AVR560 (< 4-fold). The immunohistochemistry studies with the Huh7 cell line, infectable with the HCV (JFH-1 strain), demonstrated that AVR560 inhibited the early steps of viral infection and blocked the spread of the HCV infection in tissue culture. The cytotoxicity in Huh7 and Vero-76 cell lines was mild. AVR560 proved to be a specific HCV inhibitor and exhibited no activity against other flaviviruses such as yellow fever (strain 17D), West Nile (strain NY99), and dengue (New Guinea type 2) in in vitro infection experiments. AVR560 also did not inhibit any of the tested human CYP450 isozymes (3A4, 1A2, 2C19 and 2D6). In the pharmacokinetic studies in mice, rats and dogs, favorable pharmacokinetic profiles and good oral bioavailability were observed for AV560. Further pre-clinical studies with this novel HCV inhibitor are in progress.

Activation of intrinsic immune responses and microglial phagocytosis in an ex vivo spinal cord slice culture model of West Nile virus infection.

UNLABELLED: West Nile virus (WNV) is a neurotropic flavivirus that causes significant neuroinvasive disease involving the brain and/or spinal cord. Experimental mouse models of WNV infection have established the importance of innate and adaptive immune responses in controlling the extent and severity of central nervous system (CNS) disease. However, differentiating between immune responses that are intrinsic to the CNS and those that are dependent on infiltrating inflammatory cells has proven difficult. We used a murine ex vivo spinal cord slice culture (SCSC) model to determine the innate immune processes specific to the CNS during WNV infections. By 7 days after ex vivo infection of SCSCs, the majority of neurons and a substantial percentage of astrocytes were infected with WNV, resulting in apoptotic cell death and astrogliosis. Microglia, the resident immune cells of the CNS, were activated by WNV infection, as exemplified by their amoeboid morphology, the development of filopodia and lamellipodia, and phagocytosis of WNV-infected cells and debris. Microglial cell activation was concomitant with increased expression of proinflammatory cytokines and chemokines, including CXCL10, CXCL1, CCL5, CCL3, CCL2, tumor necrosis factor alpha (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and interleukin-6 (IL-6). The application of minocycline, an inhibitor of neuroinflammation, altered the WNV-induced proinflammatory cytokine/chemokine expression profile, with inhibited production of CCL5, CCL2, and IL-6. Our findings establish that CNS-resident cells have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses. IMPORTANCE: There are no specific treatments of proven efficacy available for WNV neuroinvasive disease. A better understanding of the pathogenesis of WNV CNS infection is crucial for the rational development of novel therapies. Development of a spinal cord slice culture (SCSC) model facilitates the study of WNV pathogenesis and allows investigation of the intrinsic immune responses of the CNS. Our studies demonstrate that robust CNS innate immune responses, including microglial activation and proinflammatory cytokine/chemokine production, develop independently of contributions from the peripheral immune system and CNS-infiltrating inflammatory cells.

P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication.

In mammalian cells, proteins involved in mRNA silencing and degradation localize to discrete cytoplasmic foci called processing or P-bodies. Here we show that microscopically visible P-bodies are greatly diminished following West Nile viral infection, but the component proteins are not depleted. On the other hand, many P-body components including LSM1, GW182, DDX3, DDX6 and XRN1, but not others like DCP1a and EDC4 are recruited to the viral replication sites, as evidenced by their colocalization at perinuclear region with viral NS3. Kinetic studies suggest that the component proteins are first released from P-bodies in response to WNV infection within 12 h post-infection, followed by recruitment to the viral replication sites by 24-36 h post-infection. Silencing of the recruited proteins individually with siRNA interfered with viral replication to varying extents suggesting that the recruited proteins are required for efficient viral replication. Thus, the P-body proteins might provide novel drug targets for inhibiting viral infection.

Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes.

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses.

The infectivity of prM-containing partially mature West Nile virus does not require the activity of cellular furin-like proteases.

Cleavage of the flavivirus prM protein by a cellular furin-like protease is a hallmark of virion maturation. While this cleavage is a required step in the viral life cycle, it can be inefficient. Virions that retain uncleaved prM may be infectious. We investigated whether cleavage by furin of prM on partially mature West Nile virus (WNV) during virus entry contributes to infectivity. Using quantitative assays of WNV infection, we found that virions incorporating considerable amounts of uncleaved prM protein were insensitive to treatment of cells with a potent inhibitor of furin activity. Thus, partially mature WNV does not require furin-like proteases for infectivity.

Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G(2337) C (Met(457) Ile) in the E gene and A(6751) G (Lys(125) Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this article could be useful for monitoring the molecular consistency and quality control of vaccine strains.

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.

Development and characterization of non-glycosylated E and NS1 mutant viruses as a potential candidate vaccine for West Nile virus.

West Nile virus is an arthropod-borne flavivirus that has caused substantial morbidity and mortality to animals as well as humans since its introduction in to the New York area in 1999. Given that there are no antiviral drugs available for treatment of the disease, vaccines provide an efficacious alternative to control this disease. Herein we describe an attenuated WNV strain developed by the ablation of the glycosylation sites in the envelope (E) and non-structural 1 (NS1) proteins. This E(154S)/NS1(130A/175A/207A) strain showed modest reduction in multiplication kinetics in cell culture and small plaque phenotype compared to the parental NY99 strain yet displayed greater than a 200,000-fold attenuation for mouse neuroinvasiveness compared to the parental strain. Mice infected with 1000PFU of E(154S)/NS1(130A/175A/207A) showed undectable viremia at either two or three days post infection; nonetheless, high titer neutralizing antibodies were detected in mice inoculated with low doses of this virus and protected against lethal challenge with a 50% protective dose of 50PFU.

[Antiviral activity of aqueous extracts and polysaccharide fractions from mycelium and fruit bodies of higher fungi].

Sixty preparations of basidiomycetes (Ganoderma, Lentinus, Pleurotus, Laetiporus, Polyporus, Inonotus, Flammulina, Grifola, Trametes) were investigated with respect to their toxicity for Vero cells and antiviral activity. The antiviral activity was estimated with the use of the West Nile virus and type 2 Herpes simplex. It was shown that 11 preparations of Ganoderma, Lentinus and Pleurotus completely inhibited the infective activity in doses not lower than 1000 TCD50 (the West Nile virus) and 100 PPU (type 2 Herpes simplex). The antiviral activity of the preparations was likely due to the content of polysaccharides or their derivatives in the composition. It increased with increasing of the quantity of the total polysaccharide fraction or its concentration.