RNA viruses can hijack vertebrate microRNAs to suppress innate immunity.

Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3' non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

Immunogenicity and Efficacy of a Measles Virus-Vectored Chikungunya Vaccine in Nonhuman Primates.

BACKGROUND: Chikungunya virus (CHIKV) infection can result in chikungunya fever (CHIKF), a self-limited acute febrile illness that can progress to chronic arthralgic sequelae in a large percentage of patients. A new measles virus-vectored vaccine was developed to prevent CHIKF, and we tested it for immunogenicity and efficacy in a nonhuman primate model. METHODS: Nine cynomolgus macaques were immunized and boosted with the measles virus-vectored chikungunya vaccine or sham-vaccinated. Sera were taken at multiple times during the vaccination phase to assess antibody responses against CHIKV. Macaques were challenged with a dose of CHIKV previously shown to cause fever and viremia, and core body temperature, viremia, and blood cell and chemistry panels were monitored. RESULTS: The vaccine was well tolerated in all macaques, and all seroconverted (high neutralizing antibody [PRNT80 titers, 40-640] and enzyme-linked immunosorbent assay titers) after the boost. Furthermore, the vaccinated primates were protected against viremia, fever, elevated white blood cell counts, and CHIKF-associated cytokine changes after challenge with the virulent La Reunión CHIKV strain. CONCLUSIONS: These results further document the immunogenicity and efficacy of a measles-vectored chikungunya vaccine that shows promise in Phase I-II clinical trials. These findings are critical to human health because no vaccine to combat CHIKF is yet licensed.

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA.

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers.

Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography.

Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen, the cryo-electron microscopy (cryoEM) grid and/or the carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.

Eastern equine encephalitis in Latin America.

BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).

Chikungunya virus 3' untranslated region: adaptation to mosquitoes and a population bottleneck as major evolutionary forces.

The 3' untranslated genome region (UTR) of arthropod-borne viruses is characterized by enriched direct repeats (DRs) and stem-loop structures. Despite many years of theoretical and experimental study, on-going positive selection on the 3'UTR had never been observed in 'real-time,' and the role of the arbovirus 3'UTR remains poorly understood. We observed a lineage-specific 3'UTR sequence pattern in all available Asian lineage of the mosquito-borne alphavirus, chikungunya virus (CHIKV) (1958-2009), including complicated mutation and duplication patterns of the long DRs. Given that a longer genome is usually associated with less efficient replication, we hypothesized that the fixation of these genetic changes in the Asian lineage 3'UTR was due to their beneficial effects on adaptation to vectors or hosts. Using reverse genetic methods, we examined the functional importance of each direct repeat. Our results suggest that adaptation to mosquitoes, rather than to mammalian hosts, is a major evolutionary force on the CHIKV 3'UTR. Surprisingly, the Asian 3'UTR appeared to be inferior to its predicted ancestral sequence for replication in both mammals and mosquitoes, suggesting that its fixation in Asia was not a result of directional selection. Rather, it may have resulted from a population bottleneck during its introduction from Africa to Asia. We propose that this introduction of a 3'UTR with deletions led to genetic drift and compensatory mutations associated with the loss of structural/functional constraints, followed by two independent beneficial duplications and fixation due to positive selection. Our results provide further evidence that the limited epidemic potential of the Asian CHIKV strains resulted from founder effects that reduced its fitness for efficient transmission by mosquitoes there.

DNA vaccine initiates replication of live attenuated chikungunya virus in vitro and elicits protective immune response in mice.

BACKGROUND: Chikungunya virus (CHIKV) causes outbreaks of chikungunya fever worldwide and represents an emerging pandemic threat. Vaccine development against CHIKV has proved challenging. Currently there is no approved vaccine or specific therapy for the disease. METHODS: To develop novel experimental CHIKV vaccine, we used novel immunization DNA (iDNA) infectious clone technology, which combines the advantages of DNA and live attenuated vaccines. Here we describe an iDNA vaccine composed of plasmid DNA that encode the full-length infectious genome of live attenuated CHIKV clone 181/25 downstream from a eukaryotic promoter. The iDNA approach was designed to initiate replication of live vaccine virus from the plasmid in vitro and in vivo. RESULTS: Experimental CHIKV iDNA vaccines were prepared and evaluated in cultured cells and in mice. Transfection with 10 ng of iDNA was sufficient to initiate replication of vaccine virus in vitro. Vaccination of BALB/c mice with a single 10 µg of CHIKV iDNA plasmid resulted in seroconversion, elicitation of neutralizing antibodies, and protection from experimental challenge with a neurovirulent CHIKV. CONCLUSIONS: Live attenuated CHIKV 181/25 vaccine can be delivered in vitro and in vivo by using DNA vaccination. The iDNA approach appears to represent a promising vaccination strategy for CHIK and other alphaviral diseases.

Chikungunya vaccine candidate is highly attenuated and protects nonhuman primates against telemetrically monitored disease following a single dose.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes major epidemics of rash, fever, and debilitating arthritis. Currently, there are no vaccines or antivirals available for prevention or treatment. We therefore generated 2 live-attenuated vaccine candidates based on the insertion of a picornavirus internal ribosome entry site (IRES) sequence into the genome of CHIKV. Vaccination of cynomolgus macaques with a single dose of either vaccine produced no signs of disease but was highly immunogenic. After challenge with a subcutaneous inoculation of wild-type CHIKV, both vaccine candidates prevented the development of detectable viremia. Protected animals also exhibited no significant changes in core body temperature or cardiovascular rhythm, whereas sham-vaccinated animals showed hyperthermia, followed by sustained hypothermia, as well as significant changes in heart rate. These CHIKV/IRES vaccine candidates appear to be safe and efficacious, supporting their strong potential as human vaccines to protect against CHIKV infection and reduce transmission and further spread.

A chimeric vesiculo/alphavirus is an effective alphavirus vaccine.

While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.

Effect of Tai Chi vs Aerobic Exercise on Blood Pressure in Patients With Prehypertension: A Randomized Clinical Trial.

Importance: Prehypertension increases the risk of developing hypertension and other cardiovascular diseases. Early and effective intervention for patients with prehypertension is highly important. Objective: To assess the efficacy of Tai Chi vs aerobic exercise in patients with prehypertension. Design, Setting, and Participants: This prospective, single-blinded randomized clinical trial was conducted between July 25, 2019, and January 24, 2022, at 2 tertiary public hospitals in China. Participants included 342 adults aged 18 to 65 years with prehypertension, defined as systolic blood pressure (SBP) of 120 to 139 mm Hg and/or diastolic BP (DBP) of 80 to 89 mm Hg. Interventions: Participants were randomized in a 1:1 ratio to a Tai Chi group (n = 173) or an aerobic exercise group (n = 169). Both groups performed four 60-minute supervised sessions per week for 12 months. Main Outcomes and Measures: The primary outcome was SBP at 12 months obtained in the office setting. Secondary outcomes included SBP at 6 months and DBP at 6 and 12 months obtained in the office setting and 24-hour ambulatory BP at 12 months. Results: Of the 1189 patients screened, 342 (mean [SD] age, 49.3 [11.9] years; 166 men [48.5%] and 176 women [51.5%]) were randomized to 1 of 2 intervention groups: 173 to Tai Chi and 169 to aerobic exercise. At 12 months, the change in office SBP was significantly different between groups by -2.40 (95% CI, -4.39 to -0.41) mm Hg (P = .02), with a mean (SD) change of -7.01 (10.12) mm Hg in the Tai Chi group vs -4.61 (8.47) mm Hg in the aerobic exercise group. The analysis of office SBP at 6 months yielded similar results (-2.31 [95% CI, -3.94 to -0.67] mm Hg; P = .006). Additionally, 24-hour ambulatory SBP (-2.16 [95% CI, -3.84 to -0.47] mm Hg; P = .01) and nighttime ambulatory SBP (-4.08 [95% CI, -6.59 to -1.57] mm Hg; P = .002) were significantly reduced in the Tai Chi group compared with the aerobic exercise group. Conclusions and Relevance: In this study including patients with prehypertension, a 12-month Tai Chi intervention was more effective than aerobic exercise in reducing SBP. These findings suggest that Tai Chi may help promote the prevention of cardiovascular disease in populations with prehypertension. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR1900024368.

Attenuation of Chikungunya virus vaccine strain 181/clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein.

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.

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.

Equine Polyclonal Antibodies Prevent Acute Chikungunya Virus Infection in Mice.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen that causes chikungunya disease (CHIK); the disease is characterized by fever, muscle ache, rash, and arthralgia. This arthralgia can be debilitating and long-lasting, seriously impacting quality of life for years. Currently, there is no specific therapy available for CHIKV infection. We have developed a despeciated equine polyclonal antibody (CHIKV-EIG) treatment against CHIKV and evaluated its protective efficacy in mouse models of CHIKV infection. In immunocompromised (IFNAR-/-) mice infected with CHIKV, daily treatment for five consecutive days with CHIKV-EIG administered at 100 mg/kg starting on the day of infection prevented mortality, reduced viremia, and improved clinical condition as measured by body weight loss. These beneficial effects were seen even when treatment was delayed to 1 day after infection. In immunocompetent mice, CHIKV-EIG treatment reduced virus induced arthritis (including footpad swelling), arthralgia-associated cytokines, viremia, and tissue virus loads in a dose-dependent fashion. Collectively, these results suggest that CHIKV-EIG is effective at preventing CHIK and could be a viable candidate for further development as a treatment for human disease.

Chimeric Chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response.

Chikungunya virus (CHIKV) is an important pathogen causing outbreaks of highly debilitating and often chronic, arthralgic human disease. We have designed chimeric alphaviruses encoding CHIKV-specific structural proteins but no structural or nonstructural proteins capable of interfering with development of cellular antiviral response. These chimeras demonstrate a highly attenuated phenotype in both immunocompetent and immunocompromised (A129) mice. However, after a single vaccination, they induced protective immune response against subsequent CHIKV challenge, characterized by high titers of neutralizing antibodies. The rational design of alphavirus genomes provides a strong basis for the development of new recombinant alphaviruses with irreversible, highly attenuated, cell type-restricted phenotypes.

Design of chimeric alphaviruses with a programmed, attenuated, cell type-restricted phenotype.

The Alphavirus genus in the Togaviridae family contains a number of human and animal pathogens. The importance of alphaviruses has been strongly underappreciated; however, epidemics of chikungunya virus (CHIKV), causing millions of cases of severe and often persistent arthritis in the Indian subcontinent, have raised their profile in recent years. In spite of a continuous public health threat, to date no licensed vaccines have been developed for alphavirus infections. In this study, we have applied an accumulated knowledge about the mechanism of alphavirus replication and protein function in virus-host interactions to introduce a new approach in designing attenuated alphaviruses. These variants were constructed from genes derived from different, geographically isolated viruses. The resulting viable variants encoded CHIKV envelope and, in contrast to naturally circulating viruses, lacked the important contributors to viral pathogenesis: genes encoding proteins functioning in inhibition of cellular transcription and downregulation of the cellular antiviral response. To make these viruses incapable of transmission by mosquito vectors and to differentially regulate expression of viral structural proteins, their replication was made dependent on the internal ribosome entry sites, derived from other positive-polarity RNA (RNA(+)) viruses. The rational design of the genomes was complemented by selection procedures, which adapted viruses to replication in tissue culture and produced variants which (i) demonstrated different levels of replication and production of the individual structural proteins, (ii) efficiently induced the antiviral response in infected cells, (iii) were incapable of replication in cells of mosquito origin, and (iv) efficiently replicated in Vero cells. This modular approach to genome design is applicable for the construction of other alphaviruses with a programmed, irreversibly attenuated phenotype.

Phenotypic and Kinetic Changes of Myeloid Lineage Cells in Innate Response to Chikungunya Infection in Cynomolgus Macaques.

Chikungunya (CHIKV) is an emerging worldwide viral threat. The immune response to infection can lead to protection and convalescence or result in long-term sequelae such as arthritis. Early innate immune events during acute infection have been characterized for some cell types, but more must be elucidated with respect to cellular responses of monocytes and other myeloid lineage cells. In addition to their roles in protection and inflammation resolution, monocytes and macrophages are sites for viral replication and may also act as viral reservoirs. These cells are also found in joints postinfection, possibly playing a role in long-term CHIKV-induced pathology. We examined kinetic and phenotypic changes in myeloid lineage cells, including monocytes, in cynomolgus macaques early after experimental infection with CHIKV. We found increased proliferation of monocytes and decreased proliferation of myeloid dendritic cells early during infection, with an accompanying decrease in absolute numbers of both cell types, as well as a simultaneous increase in plasmacytoid dendritic cell number. An increase in CD16 and CD14 was seen along with a decrease in monocyte Human Leukocyte Antigen-DR isotype expression within 3 days of infection, potentially indicating monocyte deactivation. A transient decrease in T cells, B cells, and natural killer cells correlated with lymphocytopenia observed during human infections with CHIKV. CD4+ T cell proliferation decreased in blood, indicating relocation of cells to effector sites. These data indicate CHIKV influences proliferation rates and kinetics of myeloid lineage cells early during infection and may prove useful in development of therapeutics and evaluation of infection-induced pathogenesis.

Collective Asymmetric Total Syntheses of Marine Decahydroquinoline Alkaloid Lepadins A-E, H, and ent-I.

Lepadins are cis-fused decahydroquinoline (DHQ) marine alkaloids that have shown diverse biological activities and have attracted extensive synthetic interest. A new collective synthetic strategy is reported that features a green chemistry approach for constructing the common cis-fused DHQ core, which is achieved through green oxone-halide oxidation for both the aza-Achmatowicz rearrangement and the intramolecular [3 + 2] cycloaddition of nitrile oxide-alkene. Collective total syntheses of lepadins A-E and H are accomplished from the common DHQ core within 10 steps.

Venezuelan equine encephalitis vaccine with rearranged genome resists reversion and protects non-human primates from viremia after aerosol challenge.

Live-attenuated V4020 vaccine for Venezuelan equine encephalitis virus (VEEV) containing attenuating rearrangement of the virus structural genes was evaluated in a non-human primate model for immunogenicity and protective efficacy against aerosol challenge with wild-type VEEV. The genomic RNA of V4020 vaccine virus was encoded in the pMG4020 plasmid under control of the CMV promoter and contained the capsid gene downstream from the glycoprotein genes. It also included attenuating mutations from the VEE TC83 vaccine, with E2-120Arg substitution genetically engineered to prevent reversion mutations. The population of V4020 vaccine virus derived from pMG4020-transfected Vero cells was characterized by next generation sequencing (NGS) and indicated no detectable genetic reversions. Cynomolgus macaques were vaccinated with V4020 vaccine virus. After one or two vaccinations including by intramuscular route, high levels of virus-neutralizing antibodies were confirmed with no viremia or apparent adverse reactions to vaccinations. The protective effect of vaccination was evaluated using an aerosol challenge with VEEV. After challenge, macaques had no detectable viremia, demonstrating a protective effect of vaccination with live V4020 VEEV vaccine.

A five-amino-acid deletion of the eastern equine encephalitis virus capsid protein attenuates replication in mammalian systems but not in mosquito cells.

Eastern equine encephalitis virus (EEEV) is a human and veterinary pathogen that causes sporadic cases of fatal neurological disease. We previously demonstrated that the capsid protein of EEEV is a potent inhibitor of host cell gene expression and that this function maps to the amino terminus of the protein. We now identify amino acids 55 to 75, within the N terminus of the capsid, as critical for the inhibition of host cell gene expression. An analysis of stable EEEV replicons expressing mutant capsid proteins corroborated these mapping data. When deletions of 5 to 20 amino acids within this region of the capsid were introduced into infectious EEEV, the mutants exhibited delayed replication in Vero cells. However, the replication of the 5-amino-acid deletion mutant in C710 mosquito cells was not affected, suggesting that virus replication and assembly were affected in a cell-specific manner. Both 5- and 20-amino-acid deletion mutant viruses exhibited increased sensitivity to interferon (IFN) in cell culture and impaired replication and complete attenuation in mice. In summary, we have identified a region within the capsid protein of EEEV that contributes to the inhibition of host gene expression and to the protection of EEEV from the antiviral effects of IFNs. This region is also critical for EEEV pathogenesis.

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence.

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.