Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

"Submergence" of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

Hypervariable Domain of nsP3 of Eastern Equine Encephalitis Virus Is a Critical Determinant of Viral Virulence.

Eastern equine encephalitis virus (EEEV) is the most pathogenic member of the Alphavirus genus in the Togaviridae family. This virus continues to circulate in the New World and has a potential for deliberate use as a bioweapon. Despite the public health threat, to date no attenuated EEEV variants have been applied as live EEEV vaccines. Our previous studies demonstrated the critical function of the hypervariable domain (HVD) in EEEV nsP3 for the assembly of viral replication complexes (vRCs). EEEV HVD contains short linear motifs that recruit host proteins required for vRC formation and function. In this study, we developed a set of EEEV mutants that contained combinations of deletions in nsP3 HVD and clustered mutations in capsid protein, and tested the effects of these modifications on EEEV infection in vivo These mutations had cumulative negative effects on viral ability to induce meningoencephalitis. The deletions of two critical motifs, which interact with the members of cellular FXR and G3BP protein families, made EEEV cease to be neurovirulent. The additional clustered mutations in capsid protein, which affect its ability to induce transcriptional shutoff, diminished EEEV's ability to develop viremia. Most notably, despite the inability to induce detectable disease, the designed EEEV mutants remained highly immunogenic and, after a single dose, protected mice against subsequent infection with wild-type (wt) EEEV. Thus, alterations of interactions of EEEV HVD and likely HVDs of other alphaviruses with host factors represent an important direction for development of highly attenuated viruses that can be applied as live vaccines.IMPORTANCE Hypervariable domains (HVDs) of alphavirus nsP3 proteins recruit host proteins into viral replication complexes. The sets of HVD-binding host factors are specific for each alphavirus, and we have previously identified those specific for EEEV. The results of this study demonstrate that the deletions of the binding sites of the G3BP and FXR protein families in the nsP3 HVD of EEEV make the virus avirulent for mice. Mutations in the nuclear localization signal in EEEV capsid protein have an additional negative effect on viral replication in vivo Despite the inability to cause a detectable disease, the double HVD and triple HVD/capsid mutants induce high levels of neutralizing antibodies. Single immunization protects mice against infection with the highly pathogenic North American strain of EEEV. High safety, the inability to revert to wild-type phenotype, and high immunogenicity make the designed mutants attractive vaccine candidates for EEEV infection.

Cooperativity between the 3' untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3' untranslated region (3' UTR) of the virus. Three of the sites are "canonical" with all 7 seed sequence residues complimentary to miR-142-3p while one is "non-canonical" and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of the seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.

Liposome-antigen-nucleic acid complexes protect mice from lethal challenge with western and eastern equine encephalitis viruses.

Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine.

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2.

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108-119 (domain A), a subtype I/IV-specific epitope at amino acids 211-226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231-246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131-146 and 241-256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.

REVIEW PAPER: pathology of animal models of alphavirus encephalitis.

The encephalitides caused by Venezuelan (VEEV), eastern (EEEV), and western (WEEV) equine encephalitis viruses are important natural diseases of horses and humans and potential agents of biowarfare or bioterrorism. No licensed vaccines or specific therapies exist to prevent or treat human infections with VEEV, EEEV, or WEEV. Well-characterized animal models are needed to support the development of such medical countermeasures under the United States Food and Drug Administration's "Animal Rule." This review focuses on the pathological features and pathogenetic mechanisms of these alphaviral encephalitides in animal models, with an emphasis on aerosol infections. Infection of mice, nonhuman primates, and other species with VEEV, EEEV, and WEEV causes encephalitis and often death. There is great variability in the specific manifestations of disease in the different models, however. Many aspects of the disease in animal models and in humans remain to be characterized using modern methods. Especially needed is a better understanding of the fundamental mechanisms involved in 3 key phases of the pathogenesis of alphavirus encephalitis. These are the early extraneural phase, the process of neuroinvasion itself, and virus and host factors related to neurovirulence. A greater understanding of these aspects could provide avenues for the development of medical countermeasures and better establish suitable animal models of alphavirus encephalitis for testing them under the Animal Rule.

A Monovalent and Trivalent MVA-Based Vaccine Completely Protects Mice Against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenge.

Venezuelan, eastern and western equine encephalitis viruses (EEV) can cause severe disease of the central nervous system in humans, potentially leading to permanent damage or death. Yet, no licensed vaccine for human use is available to protect against these mosquito-borne pathogens, which can be aerosolized and therefore pose a bioterror threat in addition to the risk of natural outbreaks. Using the mouse aerosol challenge model, we evaluated the immunogenicity and efficacy of EEV vaccines that are based on the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform: three monovalent vaccines expressing the envelope polyproteins E3-E2-6K-E1 of the respective EEV virus, a mixture of these three monovalent EEV vaccines (Triple-Mix) as a first approach to generate a multivalent vaccine, and a true multivalent alphavirus vaccine (MVA-WEV, Trivalent) encoding the polyproteins of all three EEVs in a single non-replicating MVA viral vector. BALB/c mice were vaccinated twice in a four-week interval and samples were assessed for humoral and cellular immunogenicity. Two weeks after the second immunization, animals were exposed to aerosolized EEV. The majority of vaccinated animals exhibited VEEV, WEEV, and EEEV neutralizing antibodies two weeks post-second administration, whereby the average VEEV neutralizing antibodies induced by the monovalent and Trivalent vaccine were significantly higher compared to the Triple-Mix vaccine. The same statistical difference was observed for VEEV E1 specific T cell responses. However, all vaccinated mice developed comparable interferon gamma T cell responses to the VEEV E2 peptide pools. Complete protective efficacy as evaluated by the prevention of mortality and morbidity, lack of clinical signs and viremia, was demonstrated for the respective monovalent MVA-EEV vaccines, the Triple-Mix and the Trivalent single vector vaccine not only in the homologous VEEV Trinidad Donkey challenge model, but also against heterologous VEEV INH-9813, WEEV Fleming, and EEEV V105-00210 inhalational exposures. These EEV vaccines, based on the safe MVA vector platform, therefore represent promising human vaccine candidates. The trivalent MVA-WEV construct, which encodes antigens of all three EEVs in a single vector and can potentially protect against all three encephalitic viruses, is currently being evaluated in a human Phase 1 trial.

Western equine encephalitis virus in Saskatchewan garter snakes and leopard frogs.

Western equine encephalitis virus was isolated from two naturally infected snakes on first bleeding and from seven others at subsequent bleedings, both with and without preliminary chilling. One snake, with neither detectable virus nor serum neutralizing antibodies when first bled, developed viremia later. Viremia in garter snakes has a cyclic rhythm independent of the temperature of the environment. Virus was isolated from 6 frogs, and 50 out of 179 had detectable serum neutralizing antibodies. Infections with this virus are widely distributed in garter snakes and leopard frogs in the agricultural area of Saskatchewan.

Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein.

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have 'elite' activity with 50 and 99% effective inhibitory concentrations (EC50 and EC99) of less than 10 and 100 ng ml-1, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.

Human-Like Neutralizing Antibodies Protect Mice from Aerosol Exposure with Western Equine Encephalitis Virus.

Western equine encephalitis virus (WEEV) causes symptoms in humans ranging from mild febrile illness to life-threatening encephalitis, and no human medical countermeasures are licensed. A previous study demonstrated that immune serum from vaccinated mice protected against lethal WEEV infection, suggesting the utility of antibodies for pre- and post-exposure treatment. Here, three neutralizing and one binding human-like monoclonal antibodies were evaluated against WEEV aerosol challenge. Dose-dependent protection was observed with two antibodies administered individually, ToR69-3A2 and ToR68-2C3. In vitro neutralization was not a critical factor for protection in this murine model, as ToR69-3A2 is a strong neutralizing antibody, and ToR68-2C3 is a non-neutralizing antibody. This result highlights the importance of both neutralizing and non-neutralizing antibodies in the protection of mice from WEEV lethality.

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate.

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ∼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ∼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from -80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at -20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.

A Multiagent Alphavirus DNA Vaccine Delivered by Intramuscular Electroporation Elicits Robust and Durable Virus-Specific Immune Responses in Mice and Rabbits and Completely Protects Mice against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenges.

There remains a need for vaccines that can safely and effectively protect against the biological threat agents Venezuelan (VEEV), western (WEEV), and eastern (EEEV) equine encephalitis virus. Previously, we demonstrated that a VEEV DNA vaccine that was optimized for increased antigen expression and delivered by intramuscular (IM) electroporation (EP) elicited robust and durable virus-specific antibody responses in multiple animal species and provided complete protection against VEEV aerosol challenge in mice and nonhuman primates. Here, we performed a comparative evaluation of the immunogenicity and protective efficacy of individual optimized VEEV, WEEV, and EEEV DNA vaccines with that of a 1 : 1 : 1 mixture of these vaccines, which we have termed the 3-EEV DNA vaccine, when delivered by IM EP. The individual DNA vaccines and the 3-EEV DNA vaccine elicited robust and durable virus-specific antibody responses in mice and rabbits and completely protected mice from homologous VEEV, WEEV, and EEEV aerosol challenges. Taken together, the results from these studies demonstrate that the individual VEEV, WEEV, and EEEV DNA vaccines and the 3-EEV DNA vaccine delivered by IM EP provide an effective means of eliciting protection against lethal encephalitic alphavirus infections in a murine model and represent viable next-generation vaccine candidates that warrant further development.

Endemic eastern equine encephalitis in the Amazon region of Peru.

Eastern equine encephalitis virus (EEEV) causes severe neurologic disease in North America, but only two fatal human cases have been documented in South America. To test the hypothesis that alphavirus heterologous antibodies cross-protect, animals were vaccinated against other alphaviruses and challenged up to 3 months later with EEEV. Short-lived cross-protection was detected, even in the absence of cross-neutralizing antibodies. To assess exposure to EEEV in Peru, sera from acutely ill and healthy persons were tested for EEEV and other alphavirus antibodies, as well as for virus isolation. No EEEV was isolated from patients living in an EEEV-enzootic area, and only 2% of individuals with febrile illness had EEEV-reactive IgM. Only 3% of healthy persons from the enzootic region had EEEV-neutralizing antibodies. Our results suggest that humans are exposed but do not develop apparent infection with EEEV because of poor infectivity and/or avirulence of South American strains.

Comparison of immune responses of brown-headed cowbird and related blackbirds to west Nile and other mosquito-borne encephalitis viruses.

The rapid geographic spread of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) across the United States has stimulated interest in comparative host infection studies to delineate competent avian hosts critical for viral amplification. We compared the host competence of four taxonomically related blackbird species (Icteridae) after experimental infection with WNV and with two endemic, mosquito-borne encephalitis viruses, western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). We predicted differences in disease resistance among the blackbird species based on differences in life history, because they differ in geographic range and life history traits that include mating and breeding systems. Differences were observed among the response of these hosts to all three viruses. Red-winged Blackbirds were more susceptible to SLEV than Brewer's Blackbirds, whereas Brewer's Blackbirds were more susceptible to WEEV than Red-winged Blackbirds. In response to WNV infection, cowbirds showed the lowest mean viremias, cleared their infections faster, and showed lower antibody levels than concurrently infected species. Brown-headed Cowbirds also exhibited significantly lower viremia responses after infection with SLEV and WEEV as well as coinfection with WEEV and WNV than concurrently infected icterids. We concluded that cowbirds may be more resistant to infection to both native and introduced viruses because they experience heightened exposure to a variety of pathogens of parenting birds during the course of their parasitic life style.

Immune responses to commercial equine vaccines against equine herpesvirus-1, equine influenza virus, eastern equine encephalomyelitis, and tetanus.

Horses are commonly vaccinated to protect against pathogens which are responsible for diseases which are endemic within the general horse population, such as equine influenza virus (EIV) and equine herpesvirus-1 (EHV-1), and against a variety of diseases which are less common but which lead to greater morbidity and mortality, such as eastern equine encephalomyelitis virus (EEE) and tetanus. This study consisted of two trials which investigated the antigenicity of commercially available vaccines licensed in the USA to protect against EIV, EHV-1 respiratory disease, EHV-1 abortion, EEE and tetanus in horses. Trial I was conducted to compare serological responses to vaccines produced by three manufacturers against EIV, EHV-1 (respiratory disease), EEE, and tetanus given as multivalent preparations or as multiple vaccine courses. Trial II compared vaccines from two manufacturers licensed to protect against EHV-1 abortion, and measured EHV-1-specific interferon-gamma (IFN-gamma) mRNA production in addition to serological evidence of antigenicity. In Trial I significant differences were found between the antigenicity of different commercial vaccines that should be considered in product selection. It was difficult to identify vaccines that generate significant immune responses to respiratory viruses. The most dramatic differences in vaccine performance occurred in the case of the tetanus antigen. In Trial II both vaccines generated significant antibody responses and showed evidence of EHV-1-specific IFN-gamma mRNA responses. Overall there were wide variations in vaccine response, and the vaccines with the best responses were not produced by a single manufacturer. Differences in vaccine performance may have resulted from differences in antigen load and adjuvant formulation.

Surveys for Antibodies Against Mosquitoborne Encephalitis Viruses in California Birds, 1996-2013.

From 1996 through 2013, 54,546 individual birds comprising 152 species and 7 orders were banded, bled, and released at four study areas within California, from which 28,388 additional serum samples were collected at one or more recapture encounters. Of these, 142, 99, and 1929 birds from 41 species were positive for neutralizing antibodies against western equine encephalomyelitis virus (WEEV), St. Louis encephalitis virus (SLEV), or West Nile virus (WNV) at initial capture or recapture, respectively. Overall, 83% of the positive serum samples were collected from five species: House Finch, House Sparrow, Mourning Dove, California Quail, and Western Scrub-Jay. Temporal data supported concurrent arbovirus surveillance and documented the disappearance of birds positive for WEEV in 2008 and SLEV in 2003 and the appearance of birds positive for WNV after its invasion in 2003. Results of these serosurveys agreed well with the host selection patterns of the Culex vectors as described from bloodmeal sequencing data and indicated that transmission of WNV seemed most effective within urban areas where avian and mosquito host diversity was limited to relatively few competent species.

Macrophage activation for cytolysis of virally infected target cells.

Bone-marrow-culture-derived macrophages killed virally infected cells but not uninfected cells. This activity could be enhanced by preexposure of the macrophages to lipopolysaccharide (LPS), and/or purified interferons. The ability to kill virally infected targets was not restricted to a single cell type or virus. Comparing the ability of activators to induce activity against virally infected targets or tumor (P815) targets, it was found that much lower levels of LPS or alpha/beta-interferon were able to induce cytolytic activity for virally infected cells than were needed for tumor targets. Further, while the antitumor activity did not change significantly with an increase in the time of exposure to activating stimuli from 4 to 24 h, the activity against virally infected cells decreased dramatically with the longer exposure to stimuli.

Prevalence of eastern equine encephalitis virus antibodies among white-tailed deer populations in Maine.

During the fall of 2010, 332 deer serum samples were collected from 15 of the 16 (93.8%) Maine counties and screened for eastern equine encephalitis virus (EEEV) antibodies using plaque reduction neutralizing tests (PRNTs). The aim was to detect and map EEEV activity in the state of Maine. Forty-seven of the 332 (14.2%) sera were positive for EEEV antibodies, showing a much wider distribution of EEEV activity in Maine than previously known. The percentage of EEEV antibody-positive deer sera was ≥10% in six counties-Piscataquis (100%), Somerset (28.6%), Waldo (22.2%), Penobscot (21.7%), Kennebec (13.7%), and Sagadahoc (10%). Positive sera were detected in all the six counties (Somerset, Waldo, Penobscot, Kennebec, Cumberland, and York) that were positive in 2009, suggesting endemic EEEV activity in these counties. EEEV antibodies were not detected in sera collected in five counties-Franklin, Knox, Lincoln, Oxford, and Washington-which was either due to low sample size or lack of EEEV activity in these counties. Our data suggest higher EEEV activity in central Maine compared to southern Maine, whereas EEEV activity in Maine has historically been associated with the southern counties of York and Cumberland.