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Hundreds of cellular host factors are required to support dengue virus infection, but their identity and roles are incompletely characterized. Here, we identify human host dependency factors required for efficient dengue virus-2 (DENV2) infection of human cells. We focused on two, TTC35 and TMEM111, which we previously demonstrated to be required for yellow fever virus (YFV) infection and others subsequently showed were also required by other flaviviruses. These proteins are components of the human endoplasmic reticulum membrane protein complex (EMC), which has roles in ER-associated protein biogenesis and lipid metabolism. We report that DENV, YFV and Zika virus (ZIKV) infections were strikingly inhibited, while West Nile virus infection was unchanged, in cells that lack EMC subunit 4. Furthermore, targeted depletion of EMC subunits in live mosquitoes significantly reduced DENV2 propagation in vivo. Using a novel uncoating assay, which measures interactions between host RNA-binding proteins and incoming viral RNA, we show that EMC is required at or prior to virus uncoating. Importantly, we uncovered a second and important role for the EMC. The complex is required for viral protein accumulation in a cell line harboring a ZIKV replicon, indicating that EMC participates in the complex process of viral protein biogenesis.

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.

Laboratory rodent influenza infection models have been and continue to be a critical tool for understanding virus-host interactions during infection. The incidence of seasonal influenza infections coupled with the need for novel therapeutics and universal vaccines highlights the need to uncover novel mechanisms of pathogenesis and protection. Mouse models are extremely useful for the evaluation of influenza vaccines and provide an invaluable tool to probe the immune response. This chapter describes the technique of intranasal inoculation of male C57BL/6J mice with an H1N1 strain of influenza (A/Puerto Rico/8/1934) and methods for assessing the optimum dose for infection, viral titers in lung tissue, and severity of disease.

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group, BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

Antipyretics reduce fever following childhood vaccinations; after inactivated influenza vaccine (IIV) they might ameliorate fever and thereby decrease febrile seizure risk, but also possibly blunt the immune response. We assessed the effect of antipyretics on immune responses and fever following IIV in children ages 6 through 47 months.Over the course of three seasons, one hundred forty-two children, receiving either a single or the first of 2 recommended doses of IIV, were randomized to receive either oral acetaminophen suspension (n = 59) or placebo (n = 59) (double-blinded) or ibuprofen (n = 24) (open-label) immediately following IIV and every 4-8 h thereafter for 24 h. Blood samples were obtained at enrollment and 4 weeks following the last recommended IIV dose. Responses to IIV were assessed by hemagglutination inhibition assay (HAI). Seroprotection was defined as an HAI titer ≥1:40 and seroconversion as a titer ≥1:40 if baseline titer1:10 or four-fold rise if baseline titer ≥1:10. Participants were monitored for fever and other solicited symptoms on the day of and day following IIV.Significant differences in seroconversion and post-vaccination seroprotection were not observed between children included in the different antipyretic groups and the placebo group for the vaccine antigens included in IIV over the course of the studies. Frequencies of solicited symptoms, including fever, were similar between treatment groups and the placebo group.Significant blunting of the immune response was not observed when antipyretics were administered to young children receiving IIV. Studies with larger sample sizes are needed to definitively establish the effect of antipyretics on IIV immunogenicity.

Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s).

Summary Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular “breathing” of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in “universal” flu vaccines.

The vector competence of Australian mosquitoes for yellow fever virus (YFV) was evaluated. Infection and transmission rates in Cairns and Townsville populations of Aedes aegypti and a Brisbane strain of Ae. notoscriptus were not significantly different from a well-characterized YFV-susceptible strain of Ae. aegypti. After exposure to 10⁷·² tissue culture infectious dose (TCID₅₀)/mL of an African strain of YFV, > 70% of Ae. aegypti and Ae. notoscriptus became infected, and > 50% transmitted the virus. When exposed to 10⁶·⁷) TCID₅₀/mL of a South American strain of YFV, the highest infection (64%) and transmission (56%) rates were observed in Ae. notoscriptus. The infection and transmission rates in the Cairns Ae. aegypti were both 24%, and they were 36% and 28%, respectively, for the Townsville population. Because competent vectors are present, the limited number of travelers from endemic areas and strict vaccination requirements will influence whether YFV transmission occurs in Australia.

Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins, Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

Phylogenetic analysis of West Nile virus in North America has identified replacement of the originally introduced clade, Eastern United States (NY99), by the North American clade. In addition, the transient emergence of other clades and genetic variants has also been observed. In this study, we investigated the potential role of the mosquito in the selection of these clades and genetic variants. We determined the relative susceptibility of Culex quinquefasciatus to infection with isolates from the Eastern U.S. clade, the North American clade, and the Southeast coastal Texas clade. Although significant differences were observed in 50% oral infectious dose values between the Eastern U.S. and two attenuated North American genetic variants compared with the North American and Southeast coastal Texas clade viruses, these differences did not correlate with persistence of the genotype in nature. These results indicate that selection of these viral genotypes was independent of viral oral infectivity in the mosquito.

Candidate vaccine ChimeriVax viruses are attenuated, efficacious, safe, and highly unlikely to be transmitted by arthropod vectors. Nevertheless, concerns have been raised about the use of these vaccines because of the potential for recombination between vaccine and wild-type (WT) strains. To evaluate the vertebrate pathogenicity of such a worst-case recombinant, ChimeriVax-dengue (DEN) 4 virus was chimerized with the WT Asibi yellow fever virus. In this worst-case scenario, chimeric viruses remained fully attenuated in nonhuman primates. We therefore conclude that, even in the highly unlikely event of "virulent" backbone reversion, the safety of ChimeriVax-DEN vaccines would not be compromised.

Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. Because CHIKV is initially introduced via the skin, where γδ T cells are prevalent, we evaluated the response of these cells to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node that was associated with the production of proinflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell −/− mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling than occurred in wild-type mice, as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell −/− mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint compared to wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage. IMPORTANCE Recent epidemics, including the 2004 to 2007 outbreak and the spread of CHIKV to naive populations in the Caribbean and Central and South America with resultant cases imported into the United States, have highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identified γδ T cells as important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics.

Three species of mosquitoes (Culex pipiens quinquefasciatus, Aedes aegypti, and Ae. albopictus) collected in Texas were compared with respect to their relative susceptibility to infection with West Nile virus (WNV) strain 114. Oral infection and dissemination rates were 73% infected with 86% dissemination for Ae. aegypti, 13% infected with 100% dissemination for Ae. albopictus, and 100% infected and disseminated for Cx. p. quinquefasciatus. The oral infectious dose required to establish a 50% infection rate was also determined. All feral mosquito species were found to be susceptible to oral infection with WNV (114) in a similar range: Ae. aegypti (6.37 log(10) 50% tissue culture infectious doses [TCID(50)]/5 microL), Ae. albopictus (6.17 log(10) TCID(50)/5 microL), and Cx. p. quinquefasciatus (5.33 log(10) TCID(50)/5 microL). These data demonstrate that both Ae. aegypti and Ae. albopictus from Texas are susceptible to infection with WNV at a similar range to Cx. p. quinquefasciatus and may represent a threat of WNV transmission to humans because of host preferences.

To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

Four chimeric yellow fever (YF) 17D-dengue (DEN) candidate vaccine viruses (ChimeriVax-DEN; Acambis, Cambridge, MA) were characterized in Aedes aegypti and Ae. albopictus mosquitoes collected from Thailand. The four vaccine viruses contained the relevant prM and E genes of wild-type dengue viruses (DENV; serotypes 1-4) substituted for the equivalent genes in the YF vaccine virus (17D) backbone. Each chimera conferred protection against the homologous DENV serotype; a tetravalent mix of all four chimeras stimulates an immune response against all serotypes. Field-collected mosquitoes from Thailand were fed on blood containing each of the viruses under study and held 21 days after infection. Infection and dissemination rates were based on antigen detection in the body or head tissues, respectively. All four wild-type DENV serotypes infected and disseminated, but the candidate vaccine viruses were highly attenuated in mosquitoes with respect to infection and especially with respect to dissemination. Considering the low level viremias anticipated in humans vaccinated with these viruses, it is predicted that the risks of infection and transmission by mosquitoes in nature is minimal.

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3′3′-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo , caused up to a 10 4 -fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity.

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. Refereed/Peer-reviewed

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 DCIR's 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.

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 x 10⁶ in BHK-21 (vertebrate) cells and ∼1.05 x 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.

Background 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; Aedes albopictus. 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. Methods In this study, using a panel of CHIKV clones that varies in sensitivity to cholesterol, we investigated the possible relationship between cholesterol dependence and Ae. albopictus infectivity. Results 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. Conclusion the E1-A226V mutation probably acts at different steps of the CHIKV life cycle, affecting multiple functions of the virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that in humans causes an acute febrile illness characterized by fever, arthralgia, and rash. It is currently associated with large outbreaks in Asia, Africa, and islands of the Indian Ocean and has been introduced from these tropical regions into Europe, where local transmission has been recorded on two occasions. The underlying basis of the pathogenesis of CHIKV and related alphaviruses that produce similar symptoms remains unclear. By applying new techniques, for example, in vivo imaging in live animals and arthropods, we may improve our understanding of viral pathogenesis in vertebrates and viral replication in mosquitoes. This technical report describes the evaluation of a CHIKV?luciferase clone to visualize infection and dissemination in both Aedes aegypti and Aedes albopictus mosquitoes and mice. In mosquitoes, luciferase activity was seen at 3 and 7 days post-infection in both head and abdomens. In vivo imaging of CHIKV-luciferase was detected in mice for up to 5 days post-infection at the site of inoculation with limited dissemination to the skeletal muscle.

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.

Persistent West Nile virus (WNV) infection in the mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is associated with pathological changes in the salivary glands, including apoptotic cell death and a corresponding reduction in virus transmission over time. The vector host response to WNV infection and the molecular basis of WNV pathogenesis in Cx. quinquefasciatus was investigated using oligonucleotide microarrays designed to detect differences in the salivary gland transcriptome between WNV-infected mosquitoes and uninfected controls. Transcripts with increased abundance in infected salivary glands included those related to immunity, transcription, protein transport and degradation, amino acid and nucleotide metabolism, signal transduction, and cellular detoxification. Microarray-based analysis detected a decrease in transcript levels of a Culex inhibitor of apoptosis gene (IAP-1) and a decrease in abundance of 11 transcripts encoding salivary gland proteins. Transcript levels for an endonuclease, a proline-rich mucin, and several D7 protein family members also decreased. Transcripts with the greatest change in abundance during infection had either no similarity to sequences found in GenBank, VectorBase, and FlyBase, or were similar to sequences with uncharacterized protein products. These transcripts represent exciting targets for future analysis. Results from this study suggest that WNV infection influences transcriptional changes in an invertebrate host target tissue that may confer an advantage to the replicating virus, induce a host defense response, and alter the composition of vector saliva. The ramifications of these changes are discussed in terms of mosquito vector competence and WNV pathogenesis.

We report the construction and comparative characterization of a full-length West Nile virus (WNV) cDNA infectious clone (ic) that contains a green fluorescent protein (GFP) expression cassette fused within the viral open reading frame. Virus derived from WNV-GFP ic stably infected Culex pipiens quinquefasciatus mosquitoes at comparable rates to virus derived from the parental (non-GFP) ic. However, insertion of this GFP cassette resulted in a temporal delay in in vivo replication kinetics and significantly decreased dissemination to head tissue. Consistent with previous reports of WNV-infected mosquito midguts, focal GFP expression was observed at 3 days post-infection (dpi), with the majority of posterior midgut epithelial cells being positive by 7 dpi. GFP foci were observed in one pair of salivary glands (1/15) dissected 14 dpi. Mice exposed to WNV-GFP-infected mosquitoes developed viremia, and GFP was detected in lymph node homogenates. These data demonstrate the effectiveness of our strategy to generate a replication competent construct with increased reporter gene stability that may be used to study early events in infection.

Phylogenetic analysis of West Nile virus in North America has identified replacement of the originally introduced clade, Eastern United States (NY99), by the North American clade. In addition, the transient emergence of other clades and genetic variants has also been observed. In this study, we investigated the potential role of the mosquito in the selection of these clades and genetic variants. We determined the relative susceptibility of Culex quinquefasciatus to infection with isolates from the Eastern U.S. clade, the North American clade, and the Southeast coastal Texas clade. Although significant differences were observed in 50% oral infectious dose values between the Eastern U.S. and two attenuated North American genetic variants compared with the North American and Southeast coastal Texas clade viruses, these differences did not correlate with persistence of the genotype in nature. These results indicate that selection of these viral genotypes was independent of viral oral infectivity in the mosquito.

Arbovirus dissemination from the midgut of a vector mosquito is a critical step in facilitating virus transmission to a susceptible host. We previously characterized the genetic determinants of yellow fever virus (YFV) dissemination from the Aedes aegypti mosquito midgut using 2 genetically and phenotypically distinct strains of YFV: the wild-type, disseminating YFV Asibi strain and the attenuated, midgut-restricted YFV 17D vaccine strain. We examined the process of viral dissemination in YFV-infected Ae. aegypti by characterizing the tissue tropisms of 3 YF viruses in Ae. aegypti: Asibi, 17D, and a chimeric virus (17D/Asibi M-E) containing the Asibi membrane (M) and envelope (E) structural protein genes and 17D nonstructural genes. Ae. aegypti were infected orally, and whole, sectioned mosquitoes were evaluated for antigen distribution at 3, 7, 10, 14, and 21 days postinfection by immunohistochemical staining. Virus antigen was consistently observed in the posterior and anterior midgut, cardial epithelium, salivary glands, fat body, and nervous tissues in Asibi- and 17D/Asibi M-E-infected Ae. aegypti following 10 or 14-day extrinsic incubation, respectively. Amplification of virus in the abdominal and thoracic fat body is hypothesized to facilitate YFV infection of the Ae. aegypti salivary glands. As expected, 17D infection was generally limited to the midgut following oral infection. However, there did not appear to be a direct correlation between distribution of infection in the midgut and dissemination to the secondary tissues.

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.

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.

To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.

The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.

The recent outbreak of Chikungunya virus (CHIKV) on several islands in the Indian Ocean and in India has focused attention on this reemerging virus and highlighted the need for development of new tools to study vector-virus-host interactions. We have constructed and characterized, in cell culture, Aedes aegypti and Ae. albopictus mosquitoes, infectious cDNA clones of CHIKV using a recent isolate from La Reunion Island. Comparison of the growth kinetics and infection rates of the viral isolate CHIKV strain LR2006 OPY1 (CHIKV-LR) and a full-length infectious clone (CHIKV-LR ic) indicate that the infectious clone has retained the viral phenotypes of the original isolate. Infectious clones that express green fluorescent protein (GFP) were also produced and characterized in cell culture and in Aedes mosquitoes. The CHIKV-LR 5'GFP infected Ae. aegypti and Ae. albopictus mosquitoes at a similar rate to the original virus and to the full length infectious clone. The CHIKV-LR 3'GFP only infected Ae. albopictus mosquitoes at similar rates. The development of these authentic infectious clones will enable targeted studies of the molecular determinants of infection, pathogenesis and transmission competence by Ae. aegypti and Ae. albopictus mosquitoes.

Four chimeric yellow fever (YF) 17D-dengue (DEN) candidate vaccine viruses (ChimeriVax-DEN; Acambis, Cambridge, MA) were characterized in Aedes aegypti and Ae. albopictus mosquitoes collected from Thailand. The four vaccine viruses contained the relevant prM and E genes of wild-type dengue viruses (DENV; serotypes 1-4) substituted for the equivalent genes in the YF vaccine virus (17D) backbone. Each chimera conferred protection against the homologous DENV serotype; a tetravalent mix of all four chimeras stimulates an immune response against all serotypes. Field-collected mosquitoes from Thailand were fed on blood containing each of the viruses under study and held 21 days after infection. Infection and dissemination rates were based on antigen detection in the body or head tissues, respectively. All four wild-type DENV serotypes infected and disseminated, but the candidate vaccine viruses were highly attenuated in mosquitoes with respect to infection and especially with respect to dissemination. Considering the low level viremias anticipated in humans vaccinated with these viruses, it is predicted that the risks of infection and transmission by mosquitoes in nature is minimal.

Many mixed hardwood stands contain pole-sized white oak (Quercus alba L.) trees that are overtopped. A major decision to be made when these stands are regenerated is whether to cut the overtopped white oak or to retain them as crop trees. This study shows that growth and quality of released white oaks can be predicted from stem size, tree age, stem and crown quality, and the degree of release. Overall the study suggests that most overtopped white oaks are not good candidates for crop trees.

Stems of d.b.h. 4 inches or greater in a low quality stand in Tennessee dominated by white and scarlet oak (Quercus coccinea) were sheared in September-November 1976, chipped, and removed. Sawtimber quality trees (30) in the 37-acre area were felled separately by conventional methods. Residual trees (2-3 inches d.b.h., ht. over 4.5 ft) in some areas were injected with herbicide. One-acre plots were planted with 1+0 loblolly pine, 2+0 white pine (Pinus strobus), or 1+0 yellow poplar, or left to regenerate naturally. After 2 years, survival of all trees was good (83% or over) and average height of loblolly pine, yellow poplar and desirable natural stems (white, scarlet or black oak, Quercus velutina) was 3.3 ft, significantly different from that of white pine (1.5 ft). It is concluded that poor quality stands can be cheaply improved by this method, although release from competing vegetation may be necessary, especially in the case of white pine.

SummaryChikungunya 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.

Results from two long-term studies involving artificial regeneration of northern red and black oak suggest that the clearcut/site-prepare/plant/clean sequence is not a viable regeneration alternative for these species. Height growth of planted oaks across a broad array of sites was unacceptably slow, and in neither study did the various cleaning regimes maintain the oaks in a favorable competitive position. North. J. Appl. For. 3:114-115, Sept. 1986.

Two groups of upland hard-wood trees from the same general area were studied by 44 volunteer observers to determine the ability of the observers to visually estimate tree ages. The error of estimate for tree ages varied by the professional category of the observer, and all categoriesof observer improved with training. The error of age estimate over all categories of observer was 40.2% on the age estimation for the first group of trees. On the second group of trees, after being supplied with the correct ages for trees in the first group, the volunteer observers reducedthe overall error to 22.1%. The best individual effort was achieved by a forester whose error on the first attempt was 16.5% with a 10.7% error on his second attempt. However, for most workers, on-the-ground training is essential for reasonable accuracy in age estimation. South. J. Appl. For.13(1):40-42.

Pole sized white oaks increase in volume growth following release from overtopping trees, but the response varies by size, age and appearance of the oaks. Significant increases in epicormic sprouting, height loss by some released trees, and highly variable stem volume growth make overtopped white oak a very questionable source of future crop trees. If overtopped trees are cut, those under 8 inches dbh and less than 80 years of age produce vigorous stump sprouts and provide a source of natural regeneration.