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Chikungunya virus (CHIKV) is a reemerging arbovirus capable of causing explosive outbreaks of febrile illness, polyarthritis, and polyarthralgia, inflicting severe morbidity on affected populations. CHIKV can be genetically classified into 3 major lineages: West African (WA); East, Central, and South African (ECSA); Indian Ocean (IOL); and Asian. Additionally, the Indian Ocean (IOL) sublineage emerged within the ECSA clade and the Asian/American sublineage emerged within the Asian clade. While differences in epidemiological and pathological characteristics among outbreaks involving different CHIKV lineages and sublineages have been suggested, few targeted investigations comparing lineage virulence levels have been reported. We compared the virulence levels of CHIKV isolates representing all major lineages and sublineages in the type I interferon receptor-knockout A129 mouse model and found lineage-specific differences in virulence. We also evaluated the cross-protective efficacy of the IOL-derived, live-attenuated vaccine strain CHIKV/IRESv1 against the Asian/American CHIKV isolate YO123223 in both murine and nonhuman primate models, as well as the WA strain SH2830 in a murine model. The CHIKV/IRES vaccine provided protection both in mice and in nonhuman primate cohorts against Caribbean strain challenge and protected mice against WA challenge. Taken together, our data suggest that Asian/American CHIKV strains are less virulent than those in the Asian, ECSA, and WA lineages and that despite differences in virulence, IOL-based vaccine strains offer robust cross-protection against strains from other lineages. Further research is needed to elucidate the genetic basis for variation in CHIKV virulence in the A129 mouse model and to corroborate this variation with human pathogenicity., IMPORTANCE Chikungunya virus (CHIKV) is a reemerging human pathogen capable of causing debilitating and disfiguring polyarthritis, which can last for months to years after initial fever has resolved. There are four major genetic lineages of CHIKV, as well as two recently emerged sublineages, none of which have been evaluated for differences in virulence. Moreover, the ability of chikungunya vaccines to cross-protect against heterologous CHIKV lineages has not been explored. Therefore, we sought to compare the virulence levels among CHIKV lineages, as well as to evaluate the cross-protective efficacy of the CHIKV/IRESv1 vaccine candidate, in two different models of CHIKV infection. Our results suggest that, although significant differences in virulence were observed among CHIKV lineages, the CHIKV/IRESv1 vaccine elicits cross-lineage protective immunity. These findings provide valuable information for predicting the severity of CHIKV-associated morbidity in future outbreaks, as well as vaccine development considerations.

Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425–428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development., IMPORTANCE Analysis of a recently observed high-resolution structure of ZIKV led to a hypothesis that its unusual stability may contribute to the associated, unique disease outcomes. Here we performed a functional analysis to demonstrate that viral prM-E genes are the main determinants for the high stability of ZIKV. The extra hydrogen-bond interaction (observed in the high-resolution structure) between ZIKV E proteins did not enhance virion stability, whereas the extended loop of E protein (CD loop in domain III) was essential for ZIKV assembly. More importantly, we found that a chimeric ZIKV with DENV-2 prM-E genes and a chimeric DENV-2 with ZIKV prM-E genes were highly attenuated in A129 mice. Mice immunized with these chimeric viruses generated robust neutralizing antibody responses and were fully protected from DENV-2 and ZIKV challenge, respectively, indicating that these chimeric viruses could be further developed as vaccine candidates.

Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis -acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.

West Nile virus (WNV) is a member of the Flavivirus genus of the family Flaviviridae. This genus contains a number of arthropod-borne human pathogens, including dengue virus (DV), Japanese encephalitis virus, yellow fever virus, and tick-borne encephalitis virus (26). WNV was associated with fever and infrequent encephalitis cases in humans in Africa, the Middle East, and Europe from its discovery in 1938 through the 1990s. In 1999, WNV was first detected in New York City, and from there it rapidly spread across the United States, some regions of Canada, Mexico, and Central America. The majority of WNV infections are asymptomatic; however, a portion of infections result in West Nile fever, and a subset of infections lead to viral invasion of the central nervous system that results in encephalitis, paralysis, and meningitis, outcomes which are especially prevalent in immunocompromised and aged individuals (4). The mechanisms by which WNV causes disease are not completely understood, but studies with a hamster model of the disease suggest that following a brief peripheral replication cycle, the virus crosses the blood-brain barrier, where it infects neurons, causing cell death (47). The direct effect of WNV on neurons or an immune response to their infection may also be responsible for encephalitis and meningoencephalitis in humans (13). Pretreatment of animals with type I interferons (IFN-α/IFN-β) has been shown to block flavivirus disease (3, 23), and animals defective in the IFN response have been shown to be more susceptible to flavivirus infection (19, 28, 39). Although the precise IFN-stimulated genes (ISGs) that are responsible for preventing or controlling flavivirus infections are unknown, a number of studies have demonstrated that flavivirus-infected cells prevent IFN-induced phosphorylation of STAT molecules, which carry the signal from the ligated IFN receptor to the nucleus to activate the transcription of ISGs (1, 11, 20, 24, 25, 32, 37, 44). Interestingly, the precise mechanism by which flaviviruses alter STAT phosphorylation appears to differ among members of the genus (1, 24, 32). IFN is produced by most eukaryotic cells in response to viral infection and/or recognition of virus-associated macromolecules (known as pathogen-associated molecular patterns [PAMPs]), such as single- or double-stranded RNA (ssRNA and dsRNA, respectively). In many mammalian cell types, ligation of Toll-like receptor 3 (TLR3) to extracellular dsRNA or recognition of intracellular dsRNA by the intracellular helicase mda5 or RIG-I induces the phosphorylation and activation of the constitutively expressed IFN regulatory factor 3 (IRF3), leading to nuclear translocation and activation of transcription of the genes for IFN-β and IFN-α subtype 4 (14). Recent studies have indicated that in cell cultures, a subset of viruses appear to activate the mda5 pathway, whereas others, including the flavivirus Japanese encephalitis virus, activate the RIG-I pathway (21). In some cases, IFN-α expression has been linked to protein kinase R activation via binding of intracellular dsRNA (9). Furthermore, multiple IFN-α subtypes can also be induced by PAMP-stimulated signal transduction pathways that lead to the phosphorylation of IRF7, which can be triggered by binding of ssRNA to TLR7/8. Interestingly, IRF7 is constitutively expressed in only a subset of cells, but the IRF7 gene is an ISG, so following IFN binding, many cells can produce IRF7, permitting them to amplify the IFN signal if stimulated by ssRNA (12). For many infections, a subset of cells known as plasmacytoid dendritic cells (pDCs), which express IRF7 constitutively, have been implicated as key IFN producers (16). These cells can produce extremely high levels of IFN in response to stimulation with infectious disease agents or components thereof. Activated pDCs also produce other cytokines, notably interleukin-12 (IL-12), tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and IL-3, and chemokines, such as CCL3, CCL4, CCL5, CCL22, CCL19, and CXCL13 (2, 5, 33). However, pDCs are not the only cell type that express IRF7 constitutively. Other lymphocytes, including monocytes, B cells, and dendritic cell precursors (pDC2) (18), also express IRF7 in the resting state and are hence able to induce synthesis of IFN-α through the IRF7 pathway (reviewed in reference 22). Virus-like particles (VLPs) have been used as tools to study RNA virus infection in vitro and in vivo. VLPs consist of subgenomic replicating genomes lacking structural protein genes (replicons) that have been encapsidated by the missing structural proteins provided in trans by packaging cells. VLPs are thus able to infect cells and initiate genome replication in a manner that mimics that of normal virus, but unlike infections with normal virus, VLP infections cannot spread in the absence of trans-expressed structural proteins. In the case of both alphaviruses (35) and flaviviruses (43), VLPs have been used to identify the first cells that are infected in insect vectors of these viruses. Furthermore, for the alphavirus Venezuelan equine encephalitis virus, VLPs have been used to identify the first cells that are infected in animal models (29), and recent studies with Venezuelan equine encephalitis virus VLPs have shown that these VLPs (referred to as VRPs in the previous study) can strongly stimulate antiviral responses (46). In the studies described in this paper, we used WNV VLPs to study the early events in WNV infection in mice, demonstrating that WNV VLPs induce a rapid IFN response, resulting in very high levels of IFN-α in the serum between 8 and 24 h after either intraperitoneal (i.p.) inoculation or subcutaneous inoculation in the footpad (f.p. inoculation). Since VLPs can undergo only a single round of infection, these studies have demonstrated that the very first cells infected in an animal are capable of stimulating the production of high levels of IFN. IFN production was dependent on the replicative capacity of VLPs, since mice inoculated with UV-inactivated VLPs did not produce IFN. Immunohistochemical (IHC) detection of WNV antigen-positive cells in the popliteal lymph nodes (pLN) after f.p. inoculation with VLPs and the demonstration of high levels of IFN mRNA in pLN collected from VLP-inoculated animals implicate this lymphoid organ as a prominent source of IFN production. Finally, the finding that IRF3−/− animals produced levels of IFN similar to those of wild-type animals suggests that neither the RIG-I/mda5 nor the TLR3 pathway, known to be important for IFN-β induction, is important for this early response to WNV infection.