Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CsCl

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm³. Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm³ and a lesser amount banded at 1.22 to 1.23 g/cm³. Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either ³²PO₄ or ³H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm³) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm³), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.     

Possible Evidence for Interference with Venezuelan Equine Encephalitis Virus Vaccination of Equines by Pre-Existing Antibody to Eastern or Western Equine Encephalitis Virus, or Both

During 1971, an epizootic of Venezuelan equine encephalitis (VEE) reached the United States. Laboratory tests were performed on a large number of sick, healthy, unvaccinated, and vaccinated horses. Neutralization (N) tests in cell cultures revealed that 153 of 193 (79.3%) equines outside the state of Texas and 175 of 204 (85.8%) within Texas (82.6% overall) had detectable N antibody to VEE virus a week or more after vaccination. Twenty-six of 40 (65%) non-Texas equines and 18 of 29 (62%) Texas equines which had no detectable antibody against VEE virus a week or more after vaccination had N antibody against Eastern equine encephalitis (EEE) or Western equine encephalitis (WEE) virus or both, whereas only 50 of 153 (32.7%) non-Texas equines and 82 of 175 (46.9%) Texas equines with demonstrable N antibody against VEE also had N antibody against EEE and/or WEE virus. In vaccinated equines, significant negative correlations were found between the occurrence of antibody to VEE and antibody to EEE and/or WEE virus. These findings support the hypothesis that pre-existing antibody to EEE and/or WEE virus may modify or interfere with infection by VEE virus. The epizoologic significance of this possibility is discussed briefly.     

东方马脑炎E2蛋白原核表达及免疫活性初步研究

为构建东方马脑炎病毒E2基因原核表达载体,完成E2蛋白表达及其免疫活性研究。利用PCR方法扩增E2编码全基因,大小为1 260 bp,将酶切后目的片段连接到原核表达载体pET-30a(+)上,构建成重组质粒pET30a(+)-EEEV-E2,采用酶切和测序分析方法鉴定正确的重组质粒转化到大肠杆菌BL21中,诱导E2蛋白表达,并用SDS-PAGE电泳和Western-blotting分析和鉴定目的蛋白;最后,用纯化的E2蛋白免疫BALB/c小鼠,小鼠随机分成4组:PBS对照组、弗氏佐剂对照组、E2蛋白免疫组和E2蛋白+弗氏佐剂免疫组,每组小鼠免疫2次,两次免疫间隔时间为14天,免疫剂量均为100μL/只;小鼠初次免疫后第10天,用细胞因子ELISA试剂盒检测血清中IL-6、IL-12与TNF-α的浓度,加强免疫后第14天,用EEEV的假病毒检测血清中E2蛋白抗体的中和作用。结果表明完成了E2基因的原核表达载体pET30a(+)-E2构建和成功表达了带有His标签的E2融合蛋白,蛋白以包涵体形式存在,大小为53.0 kDa;免疫小鼠血清中产生了高水平的IL-6、IL-12与TNF-α和具有较强中和作用的E2蛋白抗体。研究结果为今后E2蛋白作为基因工程亚单位疫苗的研究提供了重要参考。     

Vector-Host Interactions of Culiseta melanura in a Focus of Eastern Equine Encephalitis Virus Activity in Southeastern Virginia

Eastern equine encephalitis virus (EEEV) causes a highly pathogenic mosquito-borne zoonosis that is responsible for sporadic outbreaks of severe illness in humans and equines in the eastern USA. Culiseta (Cs.) melanura is the primary vector of EEEV in most geographic regions but its feeding patterns on specific avian and mammalian hosts are largely unknown in the mid-Atlantic region. The objectives of our study were to: 1) identify avian hosts of Cs. melanura and evaluate their potential role in enzootic amplification of EEEV, 2) assess spatial and temporal patterns of virus activity during a season of intense virus transmission, and 3) investigate the potential role of Cs. melanura in epidemic/epizootic transmission of EEEV to humans and equines. Accordingly, we collected mosquitoes at 55 sites in Suffolk, Virginia in 2013, and identified the source of blood meals in engorged mosquitoes by nucleotide sequencing PCR products of the mitochondrial cytochrome b gene. We also examined field-collected mosquitoes for evidence of infection with EEEV using Vector Test, cell culture, and PCR. Analysis of 188 engorged Cs. melanura sampled from April through October 2013 indicated that 95.2%, 4.3%, and 0.5% obtained blood meals from avian, mammalian, and reptilian hosts, respectively. American Robin was the most frequently identified host for Cs. melanura (42.6% of blood meals) followed by Northern Cardinal (16.0%), European Starling (11.2%), Carolina Wren (4.3%), and Common Grackle (4.3%). EEEV was detected in 106 mosquito pools of Cs. melanura, and the number of virus positive pools peaked in late July with 22 positive pools and a Maximum Likelihood Estimation (MLE) infection rate of 4.46 per 1,000 mosquitoes. Our findings highlight the importance of Cs. melanura as a regional EEEV vector based on frequent feeding on virus-competent bird species. A small proportion of blood meals acquired from mammalian hosts suggests the possibility that this species may occasionally contribute to epidemic/epizootic transmission of EEEV.     

Biological, Physical, and Chemical Properties of Eastern Equine Encephalitis Virus I. Purification and Physical Properties

A new purification procedure was adopted for Eastern equine encephalitis virus which does not subject the virus to pelleting at any stage. Three- to 4-liter volumes were passed through a diethylaminoethyl cellulose column. The virus-containing fractions were banded on a sucrose cushion and finally concentrated in an isopycnic band in a linear sucrose gradient. This method reduced the volume 1,000-fold with a concomitant increase in viral titer, i.e., better than 90% recovery. Numerous criteria have been used to establish that this viral preparation was essentially free from cellular debris and nonviral material. Physical studies on this purified viral product were initiated. The sedimentation coefficient as determined by band sedimentation was 240S, the buoyant density in sucrose was 1.18 g/cc, and the diameter of the virus was 54 nm. From the diameter and the buoyant density it was possible to calculate the molecular weight of a spherical particle. In this case, the calculated molecular weight for Eastern equine encephalitis virus was 58 x 10(6) daltons.     

Temperature-sensitive Steps in the Biosynthesis of Venezuelan Equine Encephalitis Virus

In contrast to Eastern equine encephalitis virus, the replication of Venezuelan equine encephalitis (VEE) virus was strongly inhibited at 44 C in chick embryo cells. The inhibited steps were analyzed by shifting the incubating temperatures up or down, and by determining during the shifts the rate and extent of infectious ribonucleic acid (RNA) synthesis, intact virus synthesis, and formation of complement-fixing antigen or of antigen detectable by a direct fluorescent-antibody technique. The inhibition appeared to be due to two temperature-sensitive steps involved in the synthesis of VEE virus in chick embryo cells. The first step of inhibition at 44 C occurred early in virus replication and could be completely reversed simply by transferring cultures to 37 C. The inhibition appeared to take place at some point between the time when the virus entered the cell and was uncoated and the beginning of viral RNA synthesis. The second temperature-sensitive step in VEE virus synthesis was irreversible; it occurred at a point after the synthesis of viral RNA, and before the formation of virus protein measured as complement-fixing antigen or as antigen that could be stained with fluorescent antibody.     

The First Outbreak of Eastern Equine Encephalitis in Vermont: Outbreak Description and Phylogenetic Relationships of the Virus Isolate

The first known outbreak of eastern equine encephalitis (EEE) in Vermont occurred on an emu farm in Rutland County in 2011. The first isolation of EEE virus (EEEV) in Vermont (VT11) was during this outbreak. Phylogenetic analysis revealed that VT11 was most closely related to FL01, a strain from Florida isolated in 2001, which is both geographically and temporally distinct from VT11. EEEV RNA was not detected in any of the 3,905 mosquito specimens tested, and the specific vectors associated with this outbreak are undetermined.     

Ecological comparisons between Eastern Asia and North America: historical and geographical perspectives

The close floristic affinities between eastern Asia and North America have long been recognized and intensively studied, however, the ecological and biogeographical consequences of such a floristic relationship between these two remotely separated regions has been largely neglected until recently. Quantitative investigations of such relationships could greatly improve our understanding of many global and historical aspects of species diversity, vegetation dynamics, and biogeography, especially with the rapid developments of cladistic methods of phylogenetic reconstruction. All comparisons and predictions will depend on understanding past and present physical and biological processes (i.e. geological history of the continents and the evolutionary history of organisms), as well as human impacts in both regions. Both species level (life history characteristics, distribution, ecological functions) and community level (species composition, structure, endemism) variables should be investigated. Comparisons should be conducted spatially across vegetation zones and temporally through geological episodes. Emphases on the phylogeny and geological history of component taxa by analysing and synthesizing multidisciplinary data would be helpful, especially with regard to the current trends in global climate change. Diversity measured on different scales (i.e. α-, β, and g-diversity) could provide invaluable information about local-regional relationships and their ecological implications in continental or even global biodiversity patterns.     

Purification, Concentration, and Inactivation of Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis (VEE) virus was purified and concentrated by chromatography of tissue culture supernatant fluids on diethylaminoethyl-cellulose columns. Stepwise gradient elution studies indicated a broad elution pattern for the virus, with recovery occurring from 0.05 to 0.7 m NaCl. Optical density, infectivity, hemagglutination (HA), and complement fixation (CF) assays indicated that complete recovery of input virus in highly purified form was possible. Single-step elution with 0.7 m tris(hydroxymethyl)aminomethane-succinate-salt buffer resulted in a virus volume decrease of 85% with a concomitant increase in infectivity and antigenicity. Recoveries consistently equaled or exceeded 100% of the input preparations. Additional purification of column-recovered virus was obtained by sedimentation of pooled virus eluates on 50% sucrose cushions. Exposure of borate saline and 0.5% histidine suspensions of purified VEE virus preparations to 6 x 10(6) r of gamma radiation resulted in a loss of infectivity for tissue culture and a loss of lethality for weanling and suckling mice. Inactivation was an exponential function of the dosage. In contrast to infectivity, antigencity (HA and CF) of both saline and histidine preparations was retained after irradiation with doses as high as 6 x 10(6) r. Purified and irradiated VEE virus preparations have been successfully used for routine serological tests and are being evaluated as vaccines.     

Comprehensive Mapping of Common Immunodominant Epitopes in the Eastern Equine Encephalitis Virus E2 Protein Recognized by Avian Antibody Responses

Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211–226 and 331–352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11–26, 30–45 and 151–166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein.     

Dynamics of Vector-Host Interactions in Avian Communities in Four Eastern Equine Encephalitis Virus Foci in the Northeastern U.S.

Background

Eastern equine encephalitis (EEE) virus (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne zoonosis that is responsible for occasional outbreaks of severe disease in humans and equines, resulting in high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. have occurred intermittently with no apparent pattern; however, during the last decade we have witnessed recurring annual emergence where EEE virus activity had been historically rare, and expansion into northern New England where the virus had been previously unknown. In the northeastern U.S., EEE virus is maintained in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine (perching) birds in freshwater hardwood swamps. However, the identity of key avian species that serve as principal virus reservoir and amplification hosts has not been established. The efficiency with which pathogen transmission occurs within an avian community is largely determined by the relative reservoir competence of each species and by ecological factors that influence contact rates between these avian hosts and mosquito vectors.

Methodology and principle findings

Contacts between vector mosquitoes and potential avian hosts may be directly quantified by analyzing the blood meal contents of field-collected specimens. We used PCR-based molecular methods and direct sequencing of the mitochondrial cytochrome b gene for profiling of blood meals in Cs. melanura, in an effort to quantify its feeding behavior on specific vertebrate hosts, and to infer epidemiologic implications in four historic EEE virus foci in the northeastern U.S. Avian point count surveys were conducted to determine spatiotemporal host community composition. Of 1,127 blood meals successfully identified to species level, >99% of blood meals were from 65 avian hosts in 27 families and 11 orders, and only seven were from mammalian hosts representing three species. We developed an empirically informed mathematical model for EEE virus transmission using Cs. melanura abundance and preferred and non-preferred avian hosts. To our knowledge this is the first mathematical model for EEE virus, a pathogen with many potential hosts, in the northeastern U.S. We measured strong feeding preferences for a number of avian species based on the proportion of mosquito blood meals identified from these bird species in relation to their observed frequencies. These included: American Robin, Tufted Titmouse, Common Grackle, Wood Thrush, Chipping Sparrow, Black-capped Chickadee, Northern Cardinal, and Warbling Vireo. We found that these bird species, most notably Wood Thrush, play a dominant role in supporting EEE virus amplification. It is also noteworthy that the competence of some of the aforementioned avian species for EEE virus has not been established. Our findings indicate that heterogeneity induced by mosquito host preference, is a key mediator of the epizootic transmission of vector-borne pathogens.

Conclusion and significance

Detailed knowledge of the vector-host interactions of mosquito populations in nature is essential for evaluating their vectorial capacity and for assessing the role of individual vertebrates as reservoir hosts involved in the maintenance and amplification of zoonotic agents of human diseases. Our study clarifies the host associations of Cs. melanura in four EEE virus foci in the northeastern U.S., identifies vector host preferences as the most important transmission parameter, and quantifies the contribution of preference-induced contact heterogeneity to enzootic transmission. Our study identifies Wood Thrush, American Robin and a few avian species that may serve as superspreaders of EEE virus. Our study elucidates spatiotemporal host species utilization by Cs. melanura in relation to avian host community. This research provides a basis to better understand the involvement of Cs. melanura and avian hosts in the transmission and ecology of EEE virus and the risk of human infection in virus foci.     

Effect of Relative Humidity and Temperature on Airborne Venezuelan Equine Encephalitis Virus

Inactivation of airborne Venezuelan equine encephalitis (VEE) virus disseminated from liquid suspensions or from lyophilized preparations as 1- to 5-mum particles was investigated under various conditions of relative humidity and temperature in a 2,500-liter static aerosol chamber. Relative humidity ranging from 18 to 90% at 24 C and temperature ranging from -40 to 24 C had no marked effect on the biological decay rate or the recovery of viable airborne VEE virus disseminated from liquid suspensions. However, at 49 C a significant increase in the biological decay rate and decrease in aerosol recovery of the VEE virus were observed. Airborne lyophilized VEE virus was significantly affected by relative humidity. An increase in relative humidity from 20 to 90% resulted in progressive decrease in aerosol recovery of viable VEE virus. A twofold reduction in aerosol recovery of the lyophilized virus was observed at and above 29 C as compared to the lower temperatures studied. However, the differences among biological decay rates of lycphilized VEE virus were not significant within temperature range of -40 to 38 C.     

Use of Hamsters for Potency Assay of Eastern and Western Equine Encephalitis Vaccines

An antigen extinction test in hamsters is described. Comparative potency assays with guinea pigs and hamsters showed the latter to be a suitable, advantageous replacement animal in these assays.     

Mosquito-Host Interactions during and after an Outbreak of Equine Viral Encephalitis in Eastern Panama

Mosquito blood meals provide information about the feeding habits and host preference of potential arthropod-borne disease vectors. Although mosquito-borne diseases are ubiquitous in the Neotropics, few studies in this region have assessed patterns of mosquito-host interactions, especially during actual disease outbreaks. Based on collections made during and after an outbreak of equine viral encephalitis, we identified the source of 338 blood meals from 10 species of mosquitoes from Aruza Abajo, a location in Darien province in eastern Panama. A PCR based method targeting three distinct mitochondrial targets and subsequent DNA sequencing was used in an effort to delineate vector-host relationships. At Aruza Abajo, large domesticated mammals dominated the assemblage of mosquito blood meals while wild bird and mammal species represented only a small portion of the blood meal pool. Most mosquito species fed on a variety of hosts; foraging index analysis indicates that eight of nine mosquito species utilize hosts at similar proportions while a stochastic model suggests dietary overlap among species was greater than would be expected by chance. The results from our null-model analysis of mosquito diet overlap are consistent with the hypothesis that in landscapes where large domestic animals dominate the local biomass, many mosquito species show little host specificity, and feed upon hosts in proportion to their biomass, which may have implications for the role of livestocking patterns in vector-borne disease ecology.     

Castoreum and Steel Traps in Eastern North America

Historians and anthropologists have credited the invention of trapping beaver with castoreum bait and steel traps to the Indians of southeastern Canada in the 1790s. However, English trappers in Virginia used this technique before 1728, and the Indians of southeastern Canada were using it by 1756. It is suggested that the widespread adoption of castoreum and steel traps by Northeastern Indians in the 1790's was due to the collapse of the world fur market after the French Revolution.     

Molecular Determinants of Mouse Neurovirulence and Mosquito Infection for Western Equine Encephalitis Virus

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90–100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.     

华东四省天目臭蛙分类修订及分布格局

运用形态学比较和12S mt DNA序列比对,对华东4省原记载的花臭蛙Odorrana schmackeri地理种群与天目臭蛙O.tianmuii、黄岗臭蛙O.huanggangensis及花臭蛙模式产地湖北宜昌种群进行比较研究。浙江、安徽、江苏、江西(三清山)花臭蛙形态学特征与天目臭蛙相吻合。基于Kimura 2-parameter模型计算花臭蛙各地理种群与天目臭蛙、黄岗臭蛙及花臭蛙湖北宜昌种群的遗传距离,结果显示,浙江、安徽、江苏、江西(三清山、彭泽)花臭蛙与湖北宜昌种群的遗传距离为0.067~0.069,与黄岗臭蛙的遗传距离为0.027~0.029,与天目臭蛙的遗传距离仅为0~0.001。因此,长江中下游以南、江南丘陵东段安徽南部、江苏、浙江、江西东北部信江以北、鄱阳湖以东的低山丘陵地带,原记录的花臭蛙应修订为天目臭蛙;江西省东北部信江以南的武夷山区为黄岗臭蛙分布范围,花臭蛙则分布于赣江鄱阳湖以西的赣西山地。赣江以东、抚河以西以及闽东丘陵地区花臭蛙的分类归属尚待确定,华东地区花臭蛙复合体的物种分化是否与水系造成的地理阻隔相关,有待深入研究。     

Patterns of Evolutionary Divergence and Convergence in the Bushy-Tailed Woodrat (Neotoma cinerea) Across Western North America

The degree of concordance between patterns of divergence in genetic and morphological characters provides insight into the process of evolutionary diversification. We analyzed species-wide morphological variation in a broadly distributed species of small mammal, the bushy-tailed woodrat (Neotoma cinerea), for comparison against a recently developed mitochondrial phylogeny. We focused our analyses on patterns of qualitative and quantitative craniodental morphological variation throughout the distribution of this taxon. We found strong concordance between the morphology of one prominent craniodental feature (sphenopalatine vacuity) and the major mitochondrial lineages, but only minimal differentiation in overall morphometric variation between these clades. Further, in contrast to the largely east–west subdivision of mitochondrial and sphenopalatine vacuity variation, cluster analyses of morphometric variation revealed a north–south morphological subdivision irrespective of mitochondrial clade. Our combined character sets are best explained by a deep history of evolutionary divergence within this taxon accompanied or followed by broad-scale convergent or parallel evolution.