The Molecular Epidemiology and Evolution of Murray Valley Encephalitis Virus: Recent Emergence of Distinct Sub-lineages of the Dominant Genotype 1

© 2015 Williams et al. Background: Recent increased activity of the mosquito-borne Murray Valley encephalitis virus (MVEV) in Australia has renewed concerns regarding its potential to spread and cause disease. Methodology/Principal Findings: To better understand the genetic relationships between earlier and more recent circulating strains, patterns of virus movement, as well as the molecular basis of MVEV evolution, complete pre-membrane (prM) and Envelope (Env) genes were sequenced from sixty-six MVEV strains from different regions of the Australasian region, isolated over a sixty year period (1951–2011). Phylogenetic analyses indicated that, of the four recognized genotypes, only G1 and G2 are contemporary. G1 viruses were dominant over the sampling period and found across the known geographic range of MVEV. Two distinct sub-lineages of G1 were observed (1A and 1B). Although G1B strains have been isolated from across mainland Australia, Australian G1A strains have not been detected outside northwest Australia. Similarly, G2 is comprised of only Western Australian isolates from mosquitoes, suggesting G1B and G2 viruses have geographic or ecological restrictions. No evidence of recombination was found and a single amino acid substitution in the Env protein (S332G) was found to be under positive selection, while several others were found to be under directional evolution. Evolutionary analyses indicated that extant genotypes of MVEV began to diverge from a common ancestor approximately 200 years ago. G2 was the first genotype to diverge, followed by G3 and G4, and finally G1, from which subtypes G1A and G1B diverged between 1964 and 1994. Conclusions/Significance: The results of this study provides new insights into the genetic diversity and evolution of MVEV. The demonstration of co-circulation of all contemporary genetic lineages of MVEV in northwestern Australia, supports the contention that this region is the enzootic focus for this virus

Molecular, antegenic and biological studies of louping-ill virus variation in the British Isles

Louping-ill (LI) virus is a member of the tick-borne encephalitis virus serocomplex in the genus Flavivirus. LI virus infection results in a biphasic encephalomyelitic disease which has been reported in many domestic animals and man. However, the vector of the virus, the sheep tick (Ixodes ricinus), is mainly associated with upland rough grazing pasture and therefore the disease most frequently affects sheep and wild red grouse (Lagopus lagopus scoticus). A vaccine, consisting of inactivated LI virus, has been available for sheep since the 1930s but the disease persists today. LI virus is the only flavivirus known to occur in the British Isles and exists at the end of a cline of tick-borne flaviviruses which has spread westward across Asia and Europe during the past 2,000 years. The spatial and temporal characteristics of LI virus evolution remain to be defined. Previous studies investigated the variation present among LI viruses in an attempt to understand the evolution of LI virus and further the knowledge of its epidemiology. The study presented here has extended this work with the investigation of antigenic, molecular and biological properties of 43 LI viruses collected from around the British IslesAntigenic analysis using monoclonal antibodies identified two types of naturally occurring escape variants. The amino acid substitutions responsible for the alternative phenotypes were identified and shown to reside within the envelope (E) protein at residues 308 and 311. Investigation of the biological properties of the LI viruses in vitro and in vivo illustrated differences among them, some of which can be associated with genetic determinantsLouping-ill (LI) virus is a member of the tick-borne encephalitis virus serocomplex in the genus Flavivirus. LI virus infection results in a biphasic encephalomyelitic disease which has been reported in many domestic animals and man. However, the vector of the virus, the sheep tick (Ixodes ricinus), is mainly associated with upland rough grazing pasture and therefore the disease most frequently affects sheep and wild red grouse (Lagopus lagopus scoticus). A vaccine, consisting of inactivated LI virus, has been available for sheep since the 1930s but the disease persists today. LI virus is the only flavivirus known to occur in the British Isles and exists at the end of a cline of tick-borne flaviviruses which has spread westward across Asia and Europe during the past 2,000 years. The spatial and temporal characteristics of LI virus evolution remain to be defined. Previous studies investigated the variation present among LI viruses in an attempt to understand the evolution of LI virus and further the knowledge of its epidemiology. The study presented here has extended this work with the investigation of antigenic, molecular and biological properties of 43 LI viruses collected from around the British IslesLouping-ill (LI) virus is a member of the tick-borne encephalitis virus serocomplex in the genus Flavivirus. LI virus infection results in a biphasic encephalomyelitic disease which has been reported in many domestic animals and man. However, the vector of the virus, the sheep tick (Ixodes ricinus), is mainly associated with upland rough grazing pasture and therefore the disease most frequently affects sheep and wild red grouse (Lagopus lagopus scoticus). A vaccine, consisting of inactivated LI virus, has been available for sheep since the 1930s but the disease persists today. LI virus is the only flavivirus known to occur in the British Isles and exists at the end of a cline of tick-borne flaviviruses which has spread westward across Asia and Europe during the past 2,000 years. The spatial and temporal characteristics of LI virus evolution remain to be defined. Previous studies investigated the variation present among LI viruses in an attempt to understand the evolution of LI virus and further the knowledge of its epidemiology. The study presented here has extended this work with the investigation of antigenic, molecular and biological properties of 43 LI viruses collected from around the British Isles.Antigenic analysis using monoclonal antibodies identified two types of naturally occurring escape variants. The amino acid substitutions responsible for the alternative phenotypes were identified and shown to reside within the envelope (E) protein at residues 308 and 311. Investigation of the biological properties of the LI viruses in vitro and in vivo illustrated differences among them, some of which can be associated with genetic determinantsMolecular analysis of the viruses by determining the nucleotide and deduced amino acid sequence of the complete E gene or a representative fragment of the gene has enabled an extended investigation of genetic variation among LI viruses. There was a distinct correlation between genetic variation and geographic distribution, with clustering of viruses from particular areas implying the occurrence of microevolution within these regions. Grouping by geography is to be expected for tick-borne viruses with non-migratory hosts which are dispersed only sporadically into new geographic regions. The only exception to this is the Irish viruses which appear to represent two distinct virus populations existing in the same tick population.Phylogenetic analysis of the sequence data implies that the ancestral LI virus was initially introduced into Ireland and at a later date into Great Britain via Wales. The virus was then transported to Scotland from where it was dispersed throughout Scotland, the north of England and Norway. More recently the virus was probably reintroduced into Ireland and also transported to the south-west of England. The nucleotide substitution rate was estimated for the LI viruses included in this study and used to calculate the dates when viral lineages diverged. This analysis implies that LI virus was introduced into the British Isles less than 600 years ago and that the most significant dispersal from Scotland occurred 100-200 years ago. The precise mode of LI virus distribution is not known, but the recent time-scale and the pattern of dispersal implicate the involvement of man and in particular the movement of livestock along specific transport route

Molecular epidemiology of dengue viruses from complete genome sequences

The availability of the complete genetic blueprint of the dengue virus is essential in molecular epidemiological studies to uncover the role of the virus in dengue pathogenesis. During the course of this project, over two hundred complete genomes of the dengue virus were generated from clinical samples collected in three dengue-endemic Southeast Asian countries. In addition, a bioinformatics platform integrating a sequence database, sequence retrieval tools, sequence annotation data and a variety of analysis tools was developed for easy management, manipulation and analysis of dengue virus sequence data. Whereas previous studies have mostly focused on epidemiological events in the Americas and Thailand, sequence data recovered from dengue epidemics in Indonesia, Malaysia and Singapore in this study have uncovered some of the dengue virus diversity circulating in the region. The three countries appear to share similar pool of dengue viruses, with some viral lineages in sustained circulation since at least the 1970s. Sequencing of historical virus isolates prior to the 2004/2005 epidemics in Indonesia and Singapore revealed that adaptive viral evolution played little or no role in triggering those epidemics. Lastly, a method that utilised all available sequence data from Malaysia was devised to reconstruct the history of dengue virus in that country since the 1960s

Comparative Characterization of Dengue Virus Serotype 2 Isolates from a South Pacific Epidemic Sweep.

Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017

A Structural Perspective of Antibody Neutralization of Dengue Virus

The four dengue viruses: DENV) are mosquito-borne flaviviruses and are considered the world\u27s most significant arboviruses in terms of worldwide disease burden. Symptoms of dengue disease are classified into dengue fever, a mild, febrile illness, and the potentially fatal severe dengue, which can include hemorrhaging and shock. Antibody protection against DENV correlates with the production of neutralizing antibodies against the envelope: E) glycoprotein. To understand the role of antibodies in DENV infection, we sought to dissect the relationship between epitope and function. Virologic studies had identified that the most potently neutralizing antibodies are against domain III: DIII) of the E protein. We have identified five epitopes within DENV DIII. Our data suggests that the most potently neutralizing antibodies are specific for a single serotype, while cross-reactive antibodies are relatively poorly neutralizing. Additionally, we were surprised to define neutralizing epitopes that were shown to be inaccessible on the surface of the virion in cryo-electron microscopy studies. Fine epitope mapping was used to define the epitopes of a panel of existing DENV-2 antibodies. Antibodies against the lateral ridge were the most potently neutralizing antibodies and reacted only with the DENV-2 serotype. The second epitope was centered on the DIII A-strand, and antibodies against this epitope reacted with several serotypes of DENV. Several poorly neutralizing antibodies reacted to all four DENV serotypes, as well as West Nile virus, a related flavivirus, mapped to the highly conserved AB loop of DIII. We expanded our studies of DIII-specific antibodies to the DENV-1 serotype. One antibody, E106, potently neutralized the five DENV-1 strains representing the five genotypes, and bound a composite epitope of the lateral ridge and A-strand epitopes. Despite the potency of E106-mediated neutralization, a combination of structural, biophysical, virologic data suggest that potent DENV-1 neutralization by E106 is coincident with bivalent engagement of the virus. Additionally, we determined the crystal structures of E111 bound to a novel fifth CC\u27 loop epitope on domain III: DIII) of the E protein from two different DENV-1 genotypes. The available atomic models of DENV virions revealed that the E111 epitope was inaccessible, suggesting that it recognizes an uncharacterized virus conformation. While the affinity of binding between E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC\u27 loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development

Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays

Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-$_{2}$, responsible for yet more than 800 000 deaths globally. Outbreaks caused by viruses, such as SARS-CoV-$_{2}$, HIV, Ebola, influenza, and Zika, have increased over the past decade, underlining the need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides for a multiplexed, high-throughput antibody analysis. This enabled for example the identification of discriminant/diagnostic epitopes in Zika or influenza and mapping epitope evolution in natural infections versus vaccinations. In this review, we highlight synthesis platforms that facilitate fast and flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines to quickly encounter pandemic threats

Arbovirus phenotype alters transmission potential

Extrinsic and environmental factors are known to affect the transmission of arthropod-borne viruses (arboviruses), including variations in the arthropod vector populations. Differences among these factors have been associated with differential transmission and are sometimes used to control the spread of an arbovirus through a vertebrate population in an effort to prevent or disrupt an outbreak. However, diversity in intrinsic viral populations, such as genetic and phenotypic variability, is not often accounted for when considering alterations in transmission. Presented in this dissertation are four experimental studies that explore the contribution of viral intrinsic factors, especially phenotypic variability, to the transmission potential of arboviruses as judged by modeling parameters such as vectorial capacity (VC) and the basic reproductive number (R0). The overall hypothesis of this research is that phenotypic differences of arboviruses alter the transmission potential of these arboviruses by conferring fitness advantages in either the vector or the vertebrate. Further, these phenotypic differences need not be large in magnitude to affect the relative transmission potential. To investigate this hypothesis, this research determined 1) whether intrinsic viral characteristics can lead to differential transmission in a given locale, 2) whether variability of viral fitness in the mosquito vector can lead to significant differential transmission potential, 3) how our newly formulated methods from our preceding aim could aid in the explanation of a currently puzzling phenomenon in the field of arbovirology, 4) whether phenotypic differences in the vertebrate host alters the potential for transmission, and 5) how identified phenotypic differences in both the vector and vertebrate hosts could act synergistically or antagonistically to alter transmission potential of arboviruses. The research in this dissertation offers a more accurate tool for assessing transmission potential in the vector, provides a new model assessing transmission potential in the vertebrate, and provides several of the necessary steps towards a more appropriate calculation of R0. Our use of R0 based on dynamic phenotypic differences provides a framework for a more dynamic formulation of transmission models, and provides an accessible framework for output validation and reporting to public health stakeholders

A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

AbstractAn acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Δ30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host

prM-reactive antibodies reveal a role for partially mature virions in dengue virus pathogenesis

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious pr

Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector