Anopheles gambiae heat shock protein cognate 70B impedes o'nyong-nyong virus replication

Background Phylogenetic and functional analysis was conducted on an Anopheles gambiae gene, ENSANGG00000017398. Based on phylogenetic analysis, this gene belongs to the same lineage as Heat shock protein cognate 70-4 (Hsc70-4) in Drosophila. Accordingly, we propose to name this gene Heat shock protein cognate 70B (HSC70B). We previously reported that expression of HSC70B and other genes including elongation factor-1α (EF-1α) and the agglutinin attachment subunit (agglutinin) were up-regulated in o'nyong-nyong virus (ONNV)-infected female An. gambiae. Double-stranded RNA interferences have been applied to further investigate HSC70B, EF-1α and the agglutinin functions in ONNV replication in An. gambiae. Results Among these three RNAi silenced genes, only dsRNAs of HSC70B (dsHSC70B) promoted ONNV replication in adult An. gambiae compared to the control mosquitoes that were co-injected with ONNV and dsRNA of β-galactosidase (dsβ-gal). ONNV titers from mosquitoes co-injected with dsHSC70B were about 9-fold higher at 6 days post-injection (d.p.i.) as compared to the control mosquitoes. By using ONNV tagged with enhanced green fluorescent protein (ONNV-eGFP), co-injection of ONNV-eGFP with dsHSC70B also showed approximately 2 ~ 3-fold higher GFP expression rates than the controls in the head, thorax, and abdomen of the mosquito. Furthermore, co-injection of ONNV with dsHSC70B significantly reduced the lifespan of adult mosquitoes as compared with the control, co-injection of ONNV with dsβ-gal treated mosquitoes. Conclusion These results indicate that HSC70B plays important roles in homeostasis and suppression of ONNV replication in the vector, An. gambiae. Biological implications of these findings are that while mosquitoes allow ONNV to replicate in them, they also check viral titers so that ONNV infection will result in no harmful effect on mosquitoes. Therefore, mosquitoes can function as vectors of ONNV transmission to humans while ONNV infection in An. gambiae remains asymptomatic.We wish to thank Dr. K. E. Olson and B. D. Foy for his kind gift of infectious clone pONNic-Foy. This research would not have been possible without the assistance of Dr. Mabel Berois with helpful guide. This project was supported by grants R01-AI44273 from NIH/NIAID to F.H.C. D.L.V. was supported by NIH T32 A10753

Chikungunya virus adaptation to Aedes albopictus mosquitoes does not correlate with acquisition of cholesterol dependence or decreased pH threshold for fusion reaction

<p>Abstract</p> <p>Background</p> <p>Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that recently caused several large scale outbreaks/epidemics of arthritic disease in tropics of Africa, Indian Ocean basin and South-East Asia. This re-emergence event was facilitated by genetic adaptation (E1-A226V substitution) of CHIKV to a newly significant mosquito vector for this virus; <it>Aedes albopictus</it>. However, the molecular mechanism explaining the positive effect of the E1-A226V mutation on CHIKV fitness in this vector remains largely unknown. Previously we demonstrated that the E1-A226V substitution is also associated with attenuated CHIKV growth in cells depleted by cholesterol.</p> <p>Methods</p> <p>In this study, using a panel of CHIKV clones that varies in sensitivity to cholesterol, we investigated the possible relationship between cholesterol dependence and <it>Ae. albopictus </it>infectivity.</p> <p>Results</p> <p>We demonstrated that there is no clear mechanistic correlation between these two phenotypes. We also showed that the E1-A226V mutation increases the pH dependence of the CHIKV fusion reaction; however, subsequent genetic analysis failed to support an association between CHIKV dependency on lower pH, and mosquito infectivity phenotypes.</p> <p>Conclusion</p> <p>the E1-A226V mutation probably acts at different steps of the CHIKV life cycle, affecting multiple functions of the virus.</p

A novel live-attenuated vaccine candidate for mayaro Fever.

Mayaro virus (MAYV) is an emerging, mosquito-borne alphavirus that causes a dengue-like illness in many regions of South America, and which has the potential to urbanize. Because no specific treatment or vaccine is available for MAYV infection, we capitalized on an IRES-based approach to develop a live-attenuated MAYV vaccine candidate. Testing in infant, immunocompetent as well as interferon receptor-deficient mice demonstrated a high degree of attenuation, strong induction of neutralizing antibodies, and efficacy against lethal challenge. This vaccine strain was also unable to infect mosquito cells, a major safety feature for a live vaccine derived from a mosquito-borne virus. Further preclinical development of this vaccine candidate is warranted to protect against this important emerging disease

Sequential Adaptive Mutations Enhance Efficient Vector Switching by Chikungunya Virus and Its Epidemic Emergence

The adaptation of Chikungunya virus (CHIKV) to a new vector, the Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics

Novel Chikungunya Vaccine Candidate with an IRES-Based Attenuation and Host Range Alteration Mechanism

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus

Development of infectious cDNA clones of Salmonid alphavirus subtype 3

<p>Abstract</p> <p>Background</p> <p>Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed.</p> <p>Results</p> <p>Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2₁₉₇, nsP3₂₆₃and nsP3₃₂₃severely reduced infectivity, a serine to proline mutation in E2₂₀₆appeared to enhance the virus titer production.</p> <p>Conclusion</p> <p>We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.</p

Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1

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Stability of Yellow Fever Virus under Recombinatory Pressure as Compared with Chikungunya Virus

Recombination is a mechanism whereby positive sense single stranded RNA viruses exchange segments of genetic information. Recent phylogenetic analyses of naturally occurring recombinant flaviviruses have raised concerns regarding the potential for the emergence of virulent recombinants either post-vaccination or following co-infection with two distinct wild-type viruses. To characterize the conditions and sequences that favor RNA arthropod-borne virus recombination we constructed yellow fever virus (YFV) 17D recombinant crosses containing complementary deletions in the envelope protein coding sequence. These constructs were designed to strongly favor recombination, and the detection conditions were optimized to achieve high sensitivity recovery of putative recombinants. Full length recombinant YFV 17D virus was never detected under any of the experimental conditions examined, despite achieving estimated YFV replicon co-infection levels of ∼2.4×106 in BHK-21 (vertebrate) cells and ∼1.05×105 in C710 (arthropod) cells. Additionally YFV 17D superinfection resistance was observed in vertebrate and arthropod cells harboring a primary infection with wild-type YFV Asibi strain. Furthermore recombination potential was also evaluated using similarly designed chikungunya virus (CHIKV) replicons towards validation of this strategy for recombination detection. Non-homologus recombination was observed for CHIKV within the structural gene coding sequence resulting in an in-frame duplication of capsid and E3 gene. Based on these data, it is concluded that even in the unlikely event of a high level acute co-infection of two distinct YFV genomes in an arthropod or vertebrate host, the generation of viable flavivirus recombinants is extremely unlikely

West Nile Virus Genetic Diversity is Maintained during Transmission by Culex pipiens quinquefasciatus Mosquitoes

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes

Chikungunya Virus and Central Nervous System Infections in Children, India

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus best known for causing fever, rash, arthralgia, and occasional neurologic disease. By using real-time reverse transcription–PCR, we detected CHIKV in plasma samples of 8 (14%) of 58 children with suspected central nervous system infection in Bellary, India. CHIKV was also detected in the cerebrospinal fluid of 3 children