[show abstract][hide abstract] ABSTRACT: A computational approach for identification and assessment of genomic sequence variability (GeneSV) is described. For a given nucleotide sequence, GeneSV collects information about the permissible nucleotide variability (changes that potentially preserve function) observed in corresponding regions in genomic sequences, and combines it with conservation/variability results from protein sequence and structure-based analyses of evaluated protein coding regions. GeneSV was used to predict effects (functional vs. non-functional) of 37 amino acid substitutions on the NS5 polymerase (RdRp) of dengue virus type 2 (DENV-2), 36 of which are not observed in any publicly available DENV-2 sequence. 32 novel mutants with single amino acid substitutions in the RdRp were generated using a DENV-2 reverse genetics system. In 81% (26 of 32) of predictions tested, GeneSV correctly predicted viability of introduced mutations. In 4 of 5 (80%) mutants with double amino acid substitutions proximal in structure to one another GeneSV was also correct in its predictions. Predictive capabilities of the developed system were illustrated on dengue RNA virus, but described in the manuscript a general approach to characterize real or theoretically possible variations in genomic and protein sequences can be applied to any organism.
Bioinformatics and biology insights 01/2014; 8:1-16.
[show abstract][hide abstract] ABSTRACT: The resurgence in cases of neurologic disease caused by West Nile virus (WNV) in the United States in 2012 came as a surprise to the general public and to many non-arbovirus researchers. Following the introduction of WNV into the US in 1999, the number of human infections rose dramatically, peaking in 2002-03. However, cases declined from 2008-11, and it was unclear if the virus would continue to have a low-level endemic transmission pattern with occasional outbreaks, like the related flavivirus, Saint Louis encephalitis virus, or a more active pattern with annual outbreaks, including occasional years with large epidemics, like Japanese encephalitis virus. The large epidemic in 2012 suggests that the United States can expect periodic outbreaks of West Nile fever and neurologic disease in the coming years. In this paper, we consider the causes of the upsurge in WNV infections during the past year and their implications for future research and disease control measures.
[show abstract][hide abstract] ABSTRACT: Serial passaging of yellow fever virus 17D in Vero cells was employed to derive seed material for a novel inactivated vaccine, XRX-001. Two independent passaging series identified a novel lysine to arginine mutation at amino acid 160 of the envelope protein, a surface-exposed residue in structural domain I. A third passage series resulted in an isoleucine to methionine mutation at residue 113 of the NS4B protein, a central membrane spanning region of the protein which has previously been associated with Vero cell adaptation of other mosquito-borne flaviviruses. These studies confirm that flavivirus adaptation to growth in Vero cells can be mediated by structural or non-structural protein mutations.
[show abstract][hide abstract] ABSTRACT: Since the 1990's West Nile virus (WNV) has become an increasingly important public health problem and cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece, Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences, and examined the evolution of this lineage. Our studies show that this lineage has evolved over the last 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group (Beasley et al. 2004. Molecular determinants of virulence of West Nile virus in North America. Arch Virol Suppl, 35-41). This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.
Journal of General Virology 11/2012; · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41°C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.
[show abstract][hide abstract] ABSTRACT: The envelope (E) protein is composed of three domains (ED1, ED2 and ED3) with ED3 targeted by the most potent neutralizing antibodies. DENV-2 strains can be divided into six genotypes. Comparison of ED3 of representative strains of the six genotypes revealed that there are nine variable residues that are specific to a given genotype. Recombinant ED3s (rED3s) of six different DENV-2 strains representing all nine variable residues were expressed, and their reactivity against a panel of two DENV-2 type-specific and three DENV complex-reactive monoclonal antibodies (mAbs) were compared. The differences in binding affinity to the rED3s representing different DENV-2 genotypes were relatively small, with the exception of type-specific-mAb 3H5 that showed up to 10-fold differences in binding between genotypes. Overall the binding differences did not lead to detectable differences in neutralization. Based on these results, DENV-2 ED3-specific neutralizing antibodies will likely be effective against DENV-2 strains from all six genotypes.
[show abstract][hide abstract] ABSTRACT: A West Nile virus (WNV) isolate from Mexico (TM171-03) and BIRD1153, a unique genotype from Texas, have exhibited reduced murine neuroinvasive phenotypes. To determine if murine neuroinvasive capacity equates to avian virulence potential, American crow (Corvus brachyrhynchos) and house sparrows (Passer domesticus) were experimentally inoculated with representative murine neuroinvasive/non-neuroinvasive strains. In both avian species, a plaque variant from Mexico that was E-glycosylation competent produced higher viremias than an E-glycosylation-incompetent variant, indicating the potential importance of E-glycosylation for avian replication. The murine non-neuroinvasive BIRD1153 strain was significantly attenuated in American crows but not house sparrows when compared with the murine neuroinvasive Texas strain. Despite the loss of murine neuroinvasive properties of nonglycosylated variants from Mexico, our data indicate avian replication potential of these strains and that unique WNV virulence characteristics exist between murine and avian models. The implications of reduced avian replication of variants from Mexico for restricted WNV transmission in Latin America is discussed.
The American journal of tropical medicine and hygiene 10/2011; 85(4):758-67. · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.
Journal of General Virology 08/2011; 92(Pt 12):2810-20. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: A recent Phase II clinical trial has demonstrated comparable safety and immunogenicity of ChimeriVax-WN02, a chimeric West Nile virus (WNV) vaccine candidate based on yellow fever 17D, in small cohorts of healthy adults aged 18-40, 41-64 and 65-80 years. Adults ≥65 years of age are an important target population for candidate WNV vaccines as they have a high risk for severe WNV neuroinvasive disease. The apparent safety and immunogenicity of ChimeriVax-WN02 in this population indicates that further development and clinical testing are justified.
Expert Review of Vaccines 05/2011; 10(5):601-4. · 4.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: The emergence of West Nile virus (WNV) in North America in 1999 as a cause of severe neurological disease in humans, horses and birds stimulated development of vaccines for human and veterinary use, as well as polyclonal/monoclonal antibodies and other immunomodulating compounds for use as therapeutics. Although disease incidence in North America has declined since the peak epidemics in 2002-2003, the virus has continued to be annually transmitted in the Americas and to cause periodic epidemics in Europe and the Middle East. Continued transmission of the virus with human and animal disease suggests that vaccines and therapeutics for the prevention and treatment of WNV disease could be of great benefit. This article focuses on progress in development and evaluation of vaccines and immunotherapeutics for the prevention and treatment of WNV disease in humans and animals.
[show abstract][hide abstract] ABSTRACT: An intact complement system is crucial for limiting West Nile virus (WNV) dissemination. Herein, we define how complement directly restricts flavivirus infection in an antibody-independent fashion. Mannose-binding lectin (MBL) recognized N-linked glycans on the structural proteins of WNV and Dengue virus (DENV), resulting in neutralization through a C3- and C4-dependent mechanism that utilized both the canonical and bypass lectin activation pathways. For WNV, neutralization occurred with virus produced in insect cells, whereas for DENV, neutralization of insect and mammalian cell-derived virus was observed. Mechanism of action studies suggested that the MBL-dependent neutralization occurred, in part, by blocking viral fusion. Experiments in mice showed an MBL-dependent accelerated intravascular clearance of DENV or a WNV mutant with two N-linked glycans on its E protein, but not with wild-type WNV. Our studies show that MBL recognizes terminal mannose-containing carbohydrates on flaviviruses, resulting in neutralization and efficient clearance in vivo.
[show abstract][hide abstract] ABSTRACT: Site-directed mutagenesis of residues in the BC loop (residues 329-333) of the envelope (E) protein domain III in a West Nile virus (WNV) infectious clone and in plasmids encoding recombinant WNV and dengue type 2 virus domain III proteins demonstrated a critical role for residues in this loop in the function and antigenicity of the E protein. This included a strict requirement for the tyrosine at residue 329 of WNV for virus viability and E domain III folding. The absence of an equivalent residue in this region of yellow fever group viruses and most tick-borne flavivirus suggests there is an evolutionary divergence in the molecular mechanisms of domain III folding employed by different flaviviruses.
[show abstract][hide abstract] ABSTRACT: In the last 10 years new concerns have arisen about safety of the live, attenuated yellow fever (YF) 17D vaccine, in particular viscerotropic adverse events, which have a case-fatality rate of 64%. A non-replicating cell culture-based vaccine would not cause these adverse events, and potentially could be used in persons with precautions or contraindications to use of the live vaccine, including age <9 months and >60 years, egg allergy, immune suppression, and pregnancy. We developed a whole virion vaccine from the 17D strain inactivated with beta-propiolactone, and adsorbed to aluminum hydroxide. The inactivated vaccine was highly immunogenic in mice, hamsters, and cynomolgus macaques. After a single dose in hamsters and macaques, neutralizing antibody titers were similar to those elicited by the live 17D vaccine (YF-VAX, Sanofi Pasteur). After two doses of inactivated vaccine, neutralizing antibody titers in hamsters were significantly higher than after a single dose of YF-VAX [geometric mean titer (GMT) 20,480 vs. 1940, respectively (P<0.001, ANOVA)]. Hamsters given a single dose or two doses of inactivated vaccine or a single dose of YF-VAX were fully protected against hepatitis, viremia, weight loss and death after challenge with YF virus (Jimenez strain). A clinical trial of the inactivated vaccine (XRX-001) has been initiated.
[show abstract][hide abstract] ABSTRACT: West Nile virus (WNV) RNA was demonstrated in 5 (20%) of 25 urine samples collected from convalescent patients 573-2452 days (1.6-6.7 years) after WNV infection. Four of the 5 amplicons sequenced showed >99% homology to the WNV NY99 strain. These findings show that individuals with chronic symptoms after WNV infection may have persistent renal infection over several years.
The Journal of Infectious Diseases 01/2010; 201(1):2-4. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: At the present time it is estimated that the process of development of a vaccine from discovery to licensure takes approximately 18-20 years and costs in excess of US$500 million. For "routine" vaccines, the case for developing a vaccine is straightforward in terms of economics and large scale public health utilization each year. For vaccines used for biodefense and emerging diseases, the considerations are somewhat different as the vaccine may not be needed every year to control outbreaks and may be stockpiled only as a countermeasure that hopefully may never be needed. Furthermore, efficacy trials are often difficult as the natural disease may be rare or not present. Consequently, animal models will play a critical role in demonstrating efficacy. Nonetheless, the vaccine pathway still requires the same fundamental components of basic science/discovery, preclinical development, clinical trials, registration/licensure, and a plan for implementation.
[show abstract][hide abstract] ABSTRACT: The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism.
[show abstract][hide abstract] ABSTRACT: Previous studies have established the therapeutic efficacy of humanized E16 (hE16) monoclonal antibody against West Nile virus in animals. Here, we assess the potential for West Nile virus strains encoding mutations in the hE16 epitope to resist passive immunotherapy and for the selection of neutralization escape variants during hE16 treatment. Resistance to hE16 in vivo was less common than expected, because several mutations that affected neutralization in vitro did not significantly affect protection in mice. Moreover, the emergence of resistant variants after infection with fully sensitive virus occurred but was relatively rare, even in highly immunocompromised B and T cell-deficient RAG mice.
The Journal of Infectious Diseases 07/2009; 200(2):202-5. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.
[show abstract][hide abstract] ABSTRACT: West Nile virus (WNV) is one of the most widely distributed of all arboviruses, with an extensive distribution throughout Africa, the Middle East, parts of Europe and the former Soviet Union, South Asia, and Australia. Until the mid-1990s, human infections with WNV were mostly associated with a mild undifferentiated fever. However, outbreaks in Europe, Israel, and North America involving humans and animals have been associated with significant rates of neurological disease (see Hayes and Gubler, 2006 for a review). The most important event was the