Charles E. McGee

A single mutation in chikungunya virus affects vector specificity and epidemic potential.

Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005–2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1–226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA–LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Epistatic Roles of E2 Glycoprotein Mutations in Adaption of Chikungunya Virus to Aedes Albopictus and Ae. Aegypti Mosquitoes

Between 2005 and 2007 Chikungunya virus (CHIKV) caused its largest outbreak/epidemic in documented history. An unusual feature of this epidemic is the involvement of Ae. albopictus as a principal vector. Previously we have demonstrated that a single mutation E1-A226V significantly changed the ability of the virus to infect and be transmitted by this vector when expressed in the background of well characterized CHIKV strains LR2006 OPY1 and 37997. However, in the current study we demonstrate that introduction of the E1-A226V mutation into the background of an infectious clone derived from the Ag41855 strain (isolated in Uganda in 1982) does not significantly increase infectivity for Ae. albopictus. In order to elucidate the genetic determinants that affect CHIKV sensitivity to the E1-A226V mutation in Ae. albopictus, the genomes of the LR2006 OPY1 and Ag41855 strains were used for construction of chimeric viruses and viruses with a specific combination of point mutations at selected positions. Based upon the midgut infection rates of the derived viruses in Ae. albopictus and Ae. aegypti mosquitoes, a critical role of the mutations at positions E2-60 and E2-211 on vector infection was revealed. The E2-G60D mutation was an important determinant of CHIKV infectivity for both Ae. albopictus and Ae. aegypti, but only moderately modulated the effect of the E1-A226V mutation in Ae. albopictus. However, the effect of the E2-I211T mutation with respect to mosquito infections was much more specific, strongly modifying the effect of the E1-A226V mutation in Ae. albopictus. In contrast, CHIKV infectivity for Ae. aegypti was not influenced by the E2-1211T mutation. The occurrence of the E2-60G and E2-211I residues among CHIKV isolates was analyzed, revealing a high prevalence of E2-211I among strains belonging to the Eastern/Central/South African (ECSA) clade. This suggests that the E2-211I might be important for adaptation of CHIKV to some particular conditions prevalent in areas occupied by ECSA stains. These newly described determinants of CHIKV mosquito infectivity for Ae. albopictus and Ae. aegypti are of particular importance for studies aimed at the investigation of the detailed mechanisms of CHIKV adaptations to its vector species.

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

Prior Exposure to Uninfected Mosquitoes Enhances Mortality in Naturally-Transmitted West Nile Virus Infection

Background The global emergence of West Nile virus (WNV) has highlighted the importance of mosquito-borne viruses. These are inoculated in vector saliva into the vertebrate skin and circulatory system. Arthropod-borne (arbo)viruses such as WNV are transmitted to vertebrates as an infectious mosquito probes the skin for blood, depositing the virus and saliva into the skin and circulation. Growing evidence has demonstrated that arthropod, and recently mosquito, saliva can have a profound effect on pathogen transmission efficiency, pathogenesis, and disease course. A potentially important aspect of natural infections that has been ignored is that in nature vertebrates are typically exposed to the feeding of uninfected mosquitoes prior to the mosquito that transmits WNV. The possibility that pre-exposure to mosquito saliva might modulate WNV infection was explored. Principal Findings Here we report that sensitization to mosquito saliva exacerbates viral infection. Prior exposure of mice to mosquito feeding resulted in increased mortality following WNV infection. This aggravated disease course was associated with enhanced early viral replication, increased interleukin-10 expression, and elevated influx of WNV-susceptible cell types to the inoculation site. This exacerbated disease course was mimicked by passive transfer of mosquito-sensitized serum. Significance This is the first report that sensitization to arthropod saliva can exacerbate arthropod-borne infection, contrary to previous studies with parasite and bacteria infections. This research suggests that in addition to the seroreactivity of the host to virus, it is important to take into account the immune response to vector feeding.

Aedes aegypti saliva alters leukocyte recruitment and cytokine signaling by antigen-presenting cells during West Nile virus infection.

West Nile virus (WNV) is transmitted during mosquito bloodfeeding. Consequently, the first vertebrate cells to contact WNV are cells in the skin, followed by those in the draining lymph node. Macrophages and dendritic cells are critical early responders in host defense against WNV infection, not just because of their role in orchestrating the immune response, but also because of their importance as sites of early peripheral viral replication. Antigen-presenting cell (APC) signals have a profound effect on host antiviral responses and disease severity. During transmission, WNV is intimately associated with mosquito saliva. Due to the ability of mosquito saliva to affect inflammation and immune responses, and the importance of understanding early events in WNV infection, we investigated whether mosquito saliva alters APC signaling during arbovirus infection, and if alterations in cell recruitment occur when WNV infection is initiated with mosquito saliva. Accordingly, experiments were performed with cultured dendritic cells and macrophages, flow cytometry was used to characterize infiltrating cell types in the skin and lymph nodes during early infection, and real-time RT-PCR was employed to evaluate virus and cytokine levels. Our in vitro results suggest that mosquito saliva significantly decreases the expression of interferon-β and inducible nitric oxide synthase in macrophages (by as much as 50 and 70%, respectively), whilst transiently enhancing interleukin-10 (IL-10) expression. In vivo results indicate that the predominate effect of mosquito feeding is to significantly reduce the recruitment of T cells, leading the inoculation site of mice exposed to WNV alone to have up to 2.8 fold more t cells as mice infected in the presence of mosquito saliva. These shifts in cell population are associated with significantly elevated IL-10 and WNV (up to 4.0 and 10 fold, respectively) in the skin and draining lymph nodes. These results suggest that mosquito saliva dysregulates APC antiviral signaling, and reveal a possible mechanism for the observed enhancement of WNV disease mediated by mosquito saliva via a reduction of T lymphocyte and antiviral activity at the inoculation site, an elevated abundance of susceptible cell types, and a concomitant increase in immunoregulatory activity of IL-10.

Isolation and Characterization of Broad and Ultrapotent Human Monoclonal Antibodies with Therapeutic Activity against Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/ml), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development.

Substitution of Wild-Type Yellow Fever Asibi Sequences for 17D Vaccine Sequences in ChimeriVax—Dengue 4 Does Not Enhance Infection of Aedes aegypti Mosquitoes

To address concerns that a flavivirus vaccine/wild-type recombinant virus might have a high mosquito infectivity phenotype, the yellow fever virus (YFV) 17D backbone of the ChimeriVax-dengue 4 virus was replaced with the corresponding gene sequences of the virulent YFV Asibi strain. Field-collected and laboratory-colonized Aedes aegypti mosquitoes were fed on blood containing each of the viruses under investigation and held for 14 days after infection. Infection and dissemination rates were based on antigen detection in titrated body or head triturates. Our data indicate that, even in the highly unlikely event of recombination or substantial backbone reversion, virulent sequences do not enhance the transmissibility of ChimeriVax viruses. In light of the low-level viremias that have been observed after vaccination in human volunteers coupled with low mosquito infectivity, it is predicted that the risk of mosquito infection and transmission of ChimeriVax vaccine recombinant/revertant viruses in nature is minimal.

Dendritic Cell Immunoreceptor Regulates Chikungunya Virus Pathogenesis in Mice

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for recent epidemic outbreaks of debilitating disease in humans. Alphaviruses are known to interact with members of the C-type lectin receptor family of pattern recognition proteins, and given that the dendritic cell immunoreceptor (DCIR) is known to act as a negative regulator of the host inflammatory response and has previously been associated with rheumatoid arthritis, we evaluated DCIRs role in response to CHIKV infection. Although we observed an increase in the proportion of dendritic cells at the site of CHIKV infection at 24 to 36 h postinfection, these cells showed decreased cell surface DCIR, suggestive of DCIR triggering and internalization. In vitro, bone marrow-derived dendritic cells from DCIR-deficient (DCIR−/−) mice exhibited altered cytokine expression following exposure to CHIKV. DCIR−/− mice exhibited more severe disease signs than wild-type C57BL6/J mice following CHIKV infection, including a more rapid and more severe onset of virus-induced edema and enhanced weight loss. Histological examination revealed that DCIR-deficient animals exhibited increased inflammation and damage in both the fascia of the inoculated foot and the ankle joint, and DCIR deficiency skewed the CHIKV-induced cytokine response at the site of infection at multiple times postinfection. Early differences in virus-induced disease between C57BL6/J and DCIR−/− mice were independent of viral replication, while extended viral replication correlated with enhanced foot swelling and tissue inflammation and damage in DCIR−/− compared to C57BL6/J mice at 6 to 7 days postinfection. These results suggest that DCIR plays a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage.

Mutational analysis of the West Nile virus NS4B protein.

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.