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Machupo virus (MACV) is the causative agent of Bolivian hemorrhagic fever. Our previous study demonstrated that a MACV strain with a single amino acid substitution (F438I) in the transmembrane domain of glycoprotein is attenuated but genetically unstable in mice. MACV is closely related to Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. Others and our group have identified the glycoprotein to be the major viral factor determining JUNV attenuation. In this study, we tested the compatibility of the glycoprotein of the Candid#1 live-attenuated vaccine strain of JUNV in MACV replication and its ability to attenuate MACV in vivo . Recombinant MACV with the Candid#1 glycoprotein (rMACV/Cd#1-GPC) exhibited growth properties similar to those of Candid#1 and was genetically stable in vitro . In a mouse model of lethal infection, rMACV/Cd#1-GPC was fully attenuated, more immunogenic than Candid#1, and fully protective against MACV infection. Therefore, the MACV strain expressing the glycoprotein of Candid#1 is safe, genetically stable, and highly protective against MACV infection in a mouse model. IMPORTANCE Currently, there are no FDA-approved vaccines and/or treatments for Bolivian hemorrhagic fever, which is a fatal human disease caused by MACV. The development of antiviral strategies to combat viral hemorrhagic fevers, including Bolivian hemorrhagic fever, is one of the top priorities of the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Here, we demonstrate for the first time that MACV expressing glycoprotein of Candid#1 is a safe, genetically stable, highly immunogenic, and protective vaccine candidate against Bolivian hemorrhagic fever.

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence., Author summary Since the identification of West Nile virus (WNV) in North America in 1999, St. Louis encephalitis virus (SLEV) cases declined rapidly. Both viruses utilize similar avian hosts and vectors for maintenance of transmission cycles; however, they present different phenotypes in both vector and avian host. In birds, WNV develops high viremias and elicits mortality whereas SLEV has not been associated with avian virulence. West Nile viral non-structural genetic elements have been demonstrated herein to dictate higher viremias in competent avian hosts and virulence in AMCRs. In contrast, non-structural SLEV elements previously have been shown to dictate increased oral infectivity in Culex mosquitoes, likely as a compensation for the lower viremias generated by SLEV. These findings coupled with the co-circulation of WNV and SLEV in Arizona in 2015 demonstrate that pre-existing flaviviral immunity does not necessarily preclude concurrent circulation of these viruses.

We have developed a transencapsidated vaccine delivery system based on the insect virus, Flock House virus (FHV). FHV is attractive due to its small genome size, simple organization, and nonpathogenic characteristics. With the insertion of a Tobacco mosaic virus (TMV) origin of assembly (Oa), the independently replicating FHV RNA1 can be transencapsidated by TMV coat protein. In this study, we demonstrated that the Oa-adapted FHV RNA1 transencapsidation process can take place in planta, by using a bipartite plant expression vector system, where TMV coat protein is expressed by another plant virus vector, Foxtail mosaic virus (FoMV). Dual infection in the same cell by both FHV and FoMV was observed. Though an apparent classical coat protein-mediated resistance repressed FHV expression, this was overcome by delaying inoculation of the TMV coat protein vector by 3 days after FHV vector inoculation. Expression of the transgene marker in animals by these in vivo-generated transencapsidated nanoparticles was confirmed by mouse vaccination, which also showed an improved vaccine response compared to similar in vitro-produced vaccines.

St. Louis encephalitis virus (SLEV) has shown greater susceptibility to oral infectivity than West Nile virus (WNV) in Culex mosquitoes. To identify the viral genetic elements that modulate these disparate phenotypes, structural chimeras (WNV–pre-membrane [prM] and envelope [E] proteins [prME]/SLEV.IC (infectious clone) and SLEV-prME/WNV.IC) were constructed in which two of the structural proteins, the prM and E, were interchanged between viruses. Oral dose–response assessment with the chimeric/parental WNV and SLEV was performed to characterize the infection phenotypes in Culex mosquitoes by artificial blood meals. The median infectious dose required to infect 50% of Cx. quinquefasciatus with WNV was indistinguishable from that of the SLEV-prME/WNV.IC chimeric virus. Similarly, SLEV and WNV-prME/SLEV.IC virus exhibited an indistinguishable oral dose–response relationship in Cx. quinquefasciatus. Infection rates for WNV.IC and SLEV-prME/WNV.IC were significantly lower than SLEV.IC and WNV-prME/SLEV.IC infection rates. These results indicated that WNV and SLEV oral infectivities are not mediated by genetic differences within the prM and E proteins.

Flock House virus (FHV) encodes a viral polymerase supporting autonomous replication of the FHV genome which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. As FHV viral genome size is strictly limited by native FHV capsid, the goal of this study was to determine if this restriction could be eliminated by inserting a non-native viral packaging signal from Tobacco Mosaic virus (TMV) to allow for trans-encapsidation by TMV coat. FHV was adapted to express enhanced green fluorescent protein (GFP) and several FHV-GFP expressing clones were generated from which full length RNA transcripts were generated. Fluorescent microscopy was used to confirm GFP expression after FHV-GFP RNA transfection into baby hamster kidney (BHK-21) cells. The TMV origin of assembly (OA) was introduced into the most robust GFP expressing FHV construct and GFP expression was used to ensure functional FHV RNA 1 activity. RNA from the best clone, FHV-C2-GFP-OA, was then mixed with TMV coat protein and monitored for encapsidation. The production of TMV-like rod shaped nanoparticles indicated that FHV-GFP-OA RNA can be encapsidated in vitro by purified TMV coat protein. This is the first demonstration of replication-independent but specific encapsidation/packaging of the FHV genome by heterologous plant viral structural proteins while maintaining FHV genome replication capacity.

Tobacco Mosaic virus (TMV) coat protein is well known for its ability to self-assemble into supramolecular nanoparticles, either as protein discs or as rods originating from the ~300 bp genomic RNA origin-of-assembly (OA). We have utilized TMV self-assembly characteristics to create a novel Flock House virus (FHV) RNA nanoparticle. FHV encodes a viral polymerase supporting autonomous replication of the FHV genome, which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. However, FHV viral genome size is strictly limited by native FHV capsid. To determine if this packaging restriction could be eliminated, FHV was adapted to express enhanced green fluorescent protein (GFP), to allow for monitoring of functional FHV RNA activity. Then TMV OA was introduced in six 3' insertion sites, with only site one supporting functional FHV GFP expression. To create nanoparticles, FHV GFP-OA modified genomic RNA was mixed in vitro with TMV coat protein and monitored for encapsidation by agarose electrophoresis and electron microscopy. The production of TMV-like rod shaped nanoparticles indicated that modified FHV RNA can be encapsidated by purified TMV coat protein by self-assembly. This is the first demonstration of replication-independent packaging of the FHV genome by protein self-assembly.

The N-linked glycosylation motif at amino acid position 154–156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A’DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus. The NYS and NYT glycosylated viruses showed higher viral replication, and lower pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A’DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status., Author summary West Nile virus (WNV) has been responsible for the largest human encephalitis epidemics in the continental United States. Avians and Culex mosquitoes are the primary hosts for WNV natural transmission cycles. The envelope (E) protein for WNV contains a variable N-linked glycosylation motif which influences avian replication, mosquito infectivity and vector competence. WNV isolates with variable E protein glycosylation motifs that have been historically associated with human cases of disease, were selected to generate WNV glycosylation mutant viruses via reverse genetics. Replication capacity and host competence of WNV glycosylation mutant viruses were compared in vitro and in vivo in American crows, house sparrows and Culex mosquitoes. The data demonstrated that N-linked glycosylation was not as crucial for WNV transmission and host competence as previously reported. Rather the amino acid identities of the glycosylation motif were more important in dictating WNV virulence phenotypes in both avian and mosquito host.

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells., Author Summary West Nile virus (WNV) is a mosquito-borne virus that has caused outbreaks in humans in many regions of the world. Birds are the natural hosts for WNV. However, different strains of WNV cause different disease outcomes in birds. Here, we compared two WNV strains, one of which causes higher mortality and generates more virus in American crows than the other. Previous research has shown that this difference is due in large part to a difference between the two strains at a single amino acid in the NS3 gene; however, this difference does not completely explain the observed effect. Here we show that another region of the viral genome also affects disease outcomes in American crows, and changes the sensitivity of the virus to temperature when grown in bird cells. These findings help us to understand the genetic features that affect WNV infection and disease outcomes in its natural host. Detection of such features in new strains of WNV and related viruses could help to understand and predict future outbreaks.

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 presence of West Nile virus (WNV) was first documented in California, USA, during the summer of 2003, and subsequently the virus has become endemic throughout the state. Sequence analysis has demonstrated that the circulating strains are representative of the North American (WN02) genotype that has displaced the East Coast genotype (NY99). A recent study has indicated that enhanced vector competence at elevated temperatures may have played a role in the displacement of the East Coast genotype by WN02. In the current study, four WN02 strains from California, including an initial 2003 isolate (COAV997), were compared to strain NY99 in growth curve assays in mosquito and duck embryonic fibroblast (DEF) cell lines at differing, biologically relevant temperatures to assess the relative temperature sensitivities of these natural isolates. COAV997 was significantly debilitated in viral replication in DEF cells at 44 °C. Full-length sequence comparison of COAV997 against the NY99 reference strain revealed non-synonymous mutations in the envelope glycoprotein (V159A), non-structural protein 1 (NS1) (K110N) and non-structural protein 4A (NS4A) (F92L), as well as two mutations in the 3′ UTR: C→T at nt 10 772 and A→G at nt 10 851. These non-synonymous mutations were introduced into the NY99 viral backbone by site-directed mutagenesis. A mutant containing the NS1-K110N and NS4A-F92L mutations exhibited a debilitated growth phenotype in DEF cells at 44 °C, similar to that of COAV997. One explanation for the subsistence of this genotype is that COAV997 was obtained from an area of California where avian host species might not present elevated temperatures. These data indicate that the NS1 and NS4A mutations identified in some WN02 isolates could reduce thermal stability and impede replication of virus at temperatures observed in febrile avian hosts.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.

A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature.

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.

Mosquito borne pathogens are transmitted to humans via saliva during blood feeding. Mosquito saliva is a complex concoction of many secretory factors that modulate the feeding foci to enhance pathogen infection and establishment. Multiple salivary proteins/factors have been identified/characterized that enhance pathogen infection. Here, we describe, for the first time, the identification of exogenous microRNAs from mosquito saliva. MicroRNAs are short, 18–24 nucleotide, non-coding RNAs that regulate gene expression, and are generally intracellular. However, circulating miRNAs have been described from serum and saliva of humans. Exogenous miRNAs have not been reported from hematophagous arthropod saliva. We sought to identify miRNAs in the mosquito saliva and their role in Chikungunya virus (CHIKV) infection. Next generation sequencing was utilized to identify 103 exogenous miRNAs in mosquito saliva of which 31 miRNAs were previously unidentified and were designated novel. Several miRNAs that we have identified are expressed only in the CHIKV infected mosquitoes. Five of the saliva miRNAs were tested for their potential to regulated CHIKV infection, and our results demonstrate their functional role in the transmission and establishment of infection during blood feeding on the host., Author Summary Mosquito saliva contains a complex repertoire of bioactive factors that are secreted into blood feeding site, the skin. Infected mosquitoes transmit pathogens to the host during feeding via saliva. The bioactive factors in mosquito saliva are responsible for modulating host hemostasis, immune defenses and pain/itch responses, and have been implicated to enhance pathogen infection and establishment in the host. In our efforts to identify and characterize salivary immunomodulators that enhance Chikungunya virus (CHIKV) transmission, we have discovered, for the first time, exogenous microRNA in mosquito saliva. MicroRNAs (miRNAs) are short, 18–24 nucleotide, non-coding RNAs that regulate gene expression. Short non-coding RNAs were extracted from the saliva of Chikungunya virus (CHIKV) infected and uninfected Aedes aegypti and Aedes albopictus saliva, and subjected to Illumina next generation sequencing. Bioinformatic analysis revealed the presence of miRNAs in the mosquito saliva. We have also identified several novel miRNAs that are expressed only during CHIKV infection. Though the functional roles of these miRNAs are yet to be established, our in-vitro data from testing 5 miRNAs demonstrate their role in the regulation of CHIKV infection. These miRNAs may play an important role in regulating the establishment of CHIKV infection in the mammalian host during blood feeding.

To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV) three reference viruses were marked genetically by site directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was assessed experimentally by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing.Luminex® technology quantitative sequencing and quantitative RT PCR (qRT PCR) were compared in regard to specificity sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. Although Luminex® technology and quantitative sequencing provided semi quantitative or qualitative measurements a sequence specific primer extension approach using a specific reverse primer set in singleplex qRT PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutant viruses. © 2013 Elsevier B.V.