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The Lyme disease spirochete, Borrelia burgdorferi , expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, while revA is not absolutely essential for infection, deletion of revA had distinct effects on dissemination, arthritis severity, and host response.

Lyme disease, the most commonly reported vector-borne disease in the United States, results from infection with Borrelia burgdorferi. Early clinical diagnosis of this disease is largely based on the presence of an erythematous skin lesion for individuals in high-risk regions. This, however, can be confused with other illnesses including southern tick−associated rash illness (STARI), an illness that lacks a defined etiological agent or laboratory diagnostic test, and is coprevalent with Lyme disease in portions of the eastern United States. By applying an unbiased metabolomics approach with sera retrospectively obtained from well-characterized patients, we defined biochemical and diagnostic differences between early Lyme disease and STARI. Specifically, a metabolic biosignature consisting of 261 molecular features (MFs) revealed that altered N -acyl ethanolamine and primary fatty acid amide metabolism discriminated early Lyme disease from STARI. Development of classification models with the 261-MF biosignature and testing against validation samples differentiated early Lyme disease from STARI with an accuracy of 85 to 98%. These findings revealed metabolic dissimilarity between early Lyme disease and STARI, and provide a powerful and new approach to inform patient management by objectively distinguishing early Lyme disease from an illness with nearly identical symptoms.

Introduction: Erythema migrans (EM) is an annular, erythematous, expanding rash that is characteristic of early Lyme disease. In the southern United States, however, many cases of EM seem to have an etiology different from that of Lyme disease. This little-understood condition is called Southern tick-associated rash illness. Methods: With the goal of obtaining biological specimens and clinical histories from 12 to 20 STARI patients for use in etiologic research, microbiologists from the Centers for Disease Control and Prevention contacted the North Carolina Network Consortium, a statewide consortium of practice-based research networks. This article describes the methods by which the North Carolina Network Consortium successfully identified and enrolled Southern tick-associated rash illness patients into a primary care-based research protocol. Results: A total of 23 patients were enrolled, with 100% attainment of the desired specimens. After an initial lack of success, the revised protocol identified and trained physicians practicing in endemic areas for the illness, used a coordinator with 24-hour availability, recruited participants using newspaper notices and medical providers, and provided regular reminders and progress updates. Conclusions: A practice-based research network can help basic scientists identify patients and collect specimens for clinically relevant research.

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA(S226A)). Although inoculation with either BbHtrA or BbHtrA(S226A) produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.

DNA was extracted from pools of Amblyomma americanum ticks collected from vegetation at two sites in Fort Leonard Wood, Missouri and tested for the presence of Borrelia spp. Two new methods were developed to detect Borrelia lonestari DNA by targeting the glycerophosphodiester phosphodiesterase (glpQ) gene. The first method detected B. lonestari DNA using a SYBR green I melting curve analysis of the PCR product obtained with glpQ gene primers. The second method, a glpQ TaqMan assay, detected and confirmed the presence of B. lonestari glpQ-specific sequences. Twenty-two of 95 tick pools collected at site A148 contained B. lonestari DNA. None of 19 pools from site A241 contained B. lonestari DNA. No B. burgdorferi sensu lato DNA was detected using a SYBR green I melting curve analysis of the PCR product obtained with outer surface protein A (ospA) primers. The overall B. lonestari infection prevalence (with 95% confidence interval) at site A148 was estimated using two algorithms: minimum infection rate 4.14% (2.45, 5.84) and maximum likelihood with correction 4.82% (3.11, 7.16). The merits of each are discussed. Sequencing of the entire B. lonestari glpQ and partial 16S rRNA genes revealed two genetic variants circulating in this population of A. americanum from Missouri.

We have generated a panel of IgG monoclonal antibodies (MAbs) directed against Borrelia burgdorferi strain B31 antigens, using a method whereby mice were primed with organisms naturally inoculated by Ixodes scapularis nymphal ticks. Western blot analysis showed that these MAbs recognized several B. burgdorferi B31 antigens, including the complement inhibitor factor H-binding proteins ErpA/I/N and ErpC. Two other MAbs were specific for the RevA protein, and have enabled characterization of that previously unknown protein. The data presented here suggest that the production of MAbs from animals infected by tick-bite is a potentially useful tool for the identification of novel proteins synthesized by B. burgdorferi during mammalian infection.

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Borrelia burgdorferiis transmitted in an enzootic cycle in Colorado between the tick Ixodes spinipalpis and the woodrat Neotoma mexicana. The genetic relationship of Colorado isolates to other B. burgdorferi isolates is unknown nor have relationships among various Colorado isolates been determined. Portions of the flagellin ( fla ), 66- kD protein, and outer surface protein A (ospA) genes were amplified from 71 Colorado isolates, screened for genetic variability using single strand conformation polymorphism analysis, and unique alleles were sequenced. Colorado isolates were most similar to tick isolates from California and New York isolate 25015. Genetic distances among Colorado ospA sequences were the same or higher than distances among other isolates whereas distances among fla sequences tended to be the same or lower. The index of association (I A ) was calculated among all loci as a measure of clonality. The IA among Colorado isolates was similar to IA previously estimated among other United States isolates. Borrelia burgdorferi sensu lato is a genetically diverse species complex consisting of four genospecies. 1-6 A gen- ospecies is a bacterial subspecies defined by degree of DNA relatedness as assessed by nucleotide sequence data or DNA- DNA reassociation. Three of these genospecies, B. burgdor- feri ss, B. garinii, and B. afzelii, cause Lyme disease and related disorders in humans and are distributed throughout Europe and Eurasia. Borrelia japonica, the fourth genospe- cies, appears to be nonpathogenic and is restricted to rodents and ticks in Asia. 2, 5 Borrelia burgdorferi ss is maintained in a diversity of enzootic cycles in the United States involving ticks of the genus Ixodes and rodent or lagomorph reser- voirs. 7-10 In California where indigenous cases of Lyme dis- ease occur, Ixodes pacificusacts as a vector of B. burgdorferi to humans 11 and is maintained in enzootic cycles primarily involving the dusky-footed wood rat (Neotoma fucipes). 7, 12 10 No known 13 Colorado isolates were identified phylogenetically as B. burgdorferi ss. We demonstrated large difference in the frequencies and diver- sity of alleles in cultured spirochetes as compared with those that were amplified from infected ticks. This observation suggested that culturing in Barbour-Stoenner-Kelley-H (BSK)-H medium selects for specific B. burgdorferi geno- types in Colorado and may underestimate the diversity of genotypes in this and potentially other enzootic cycles. Of greater concern, these results suggest the possibility that the prevalence of B. burgdorferi may be underestimated in cer- tain enzootic cycles in the United States if they also involve genotypes that cannot be cultured. The primary goal of this study was to perform a phylogenetic analysis of each of the alleles at the three loci to estimate the relationship of Col- orado isolates relative to other B. burgdorferi ss isolates. In addition, we compare genetic diversity among Colorado iso- lates relative to the diversity among other B. burgdorferi isolates. The rate with which selection can act on a set of organisms is proportional to the genetic variance among those organisms.

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.

To examine in detail Borrelia burgdorferi strain diversity in the United States, 186 isolates from human, tick, and rodent sources were analyzed from multiple distinct geographic regions of the United States and abroad. Strains were characterized by genomic macrorestriction analysis and ospA and 23S rDNA gene sequencing followed by phylogenetic analysis. Results indicate that spirochetal isolates from the United States fall into two major divisions and nine or more subdivisions; human isolates fell into five of these subdivisions. Greater genetic diversity was observed among B. burgdorferi isolates from moderate climatic regions, consistent with increased tick vector and reservoir diversity. All of the Borrelia isolates were reactive by ospA polymerase chain reaction except for Borrelia hermsii controls and several tick isolates from the Northeast, which were shown to lack the 49-kb plasmid encoding outer surface protein A (OspA). The data suggest that US B. burgdorferi isolates demonstrate substantial genetic heterogeneity, with regional differences in spirochete populations.

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing

Polymerase chain reaction primers specific for Ehrlichia chaffeensis were used to amplify DNA from extracts of pooled ticks. Amplification was performed on extracts from 140 pools (1,579 total ticks) consisting of three tick genera collected from five states. The characteristic 389-basepair product was observed after amplification of extracts from seven different pools of adult Amblyomma americanum (117 pools, 1,462 ticks), but not from pools of nymphs. No specific product was observed after amplification of 20 pools (105 ticks) of Dermacentor variabilis and three pools of Ixodes scapularis (12 ticks). Ehrlichia chaffeensis was present in A. americanum at a minimum frequency of > or = 0.48%, suggesting that A. americanum may be a vector of human ehrlichiosis.

The causative agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes ricinus complex. In this study, we report the antibody response of recombinant inbred strains of mice of the H-2, b, d, and k haplotypes, infected with B. burgdorferi as a result of exposure to infected I. dammini. The patterns of antibody response assayed by Western blot analysis indicate significant major histocompatibility complex (MHC) restriction to bacterial antigens within the first 2 mo of infection in mice. Other bacterial antigens induce a significant response across the MHC haplotypes tested when assayed on the same bacterial strain used to transmit the infection, but do not crossreact with the same proteins derived from heterologous strains of B. burgdorferi. No response to outer surface protein A was detected at any time during the 60-d period we analyzed this infection. A third group of bacterial antigens appear to generate a MHC-nonrestricted response, and this lack of restriction is maintained when assaying the crossreactivity of the response with other strains of B. burgdorferi. These proteins may provide more accurate diagnostic probes than those currently in use. Finally, there appears to be a significant difference in the expression of most bacterial antigens when the spirochete is cultured for many passages since the same strain of bacterium isolated from low-passage and high-passage preparations exhibit different banding patterns in Western blots when assayed with the same sera.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.

Stable neutralization (N) escape variants of Venezuelan equine encephalitis (VEE) virus were selected by anti-E2 glycoprotein monoclonal antibodies (MAbs) that neutralize viral infectivity, block viral hemagglutination, and passively protect mice. The nucleotide sequence of the E1, E2, and E3 genes of four variants revealed a clustering of single mutations in a domain spanning E2-182 to E2-207. The conformation of this short linear sequence affects antigenicity in the N domain because reduction and alkylation of virus disrupted binding of some E2 neutralizing MAbs. Serologic evidence for interaction of E2 epitopes also was obtained. Mutations in the N domain of VEE virus did not alter the kinetics of binding to Vero cells. They did, in some cases, produce attenuation of virulence in mice.

Heat shock protein 10 (Hsp10) and heat shock protein 60 (Hsp60) were originally described as essential mitochondrial proteins involved in protein folding. However, both proteins have also been shown to have a number of extracellular immunomodulatory activities. Here we show that purified recombinant human Hsp10 incubated with cells in vitro reduced lipopolysaccharide (LPS)-induced nuclear factor-kappaB activation and secretion of several inflammatory mediators from RAW264.7 cells, murine macrophages, and human peripheral blood mononuclear cells. Induction of tolerance by contaminating LPS was formally excluded as being responsible for Hsp10 activity. Treatment of mice with Hsp10 before endotoxin challenge resulted in the reduction of serum tumor necrosis factor-alpha and RANTES (regulated upon activation, normal T cell expressed and secreted) levels and an elevation of serum interleukin-10 levels. Hsp10 treatment also delayed mortality in a murine graft-versus-host disease model, where gut-derived LPS contributes to pathology. We were unable to confirm previous reports that Hsp10 has tumor growth factor properties and suggest that Hsp10 exerts anti-inflammatory activity by inhibiting Toll-like receptor signaling possibly by interacting with extracellular Hsp60.

A glpQ ortholog was identified in DNA from Borrelia lonestari-positive Amblyomma americanum, providing further evidence that B. lonestari is more closely related to the relapsing fever group spirochetes than to borreliae that cause Lyme disease. This finding provides a basis for developing diagnostic assays to differentiate species of borrelia transmitted by hard ticks.

Amblyomma americanum collected near Lake Wappapello, Missouri, tested positive for Borrelia lonestari using polymerase chain reaction and sequence analyses of B. lonestari 16S rRNA and flagellin (flaB) genes. Twelve pools containing a total of 214 nymph or adult ticks contained evidence of infection with B. lonestari (minimum prevalence 5.6%). These data suggest that persons in southeast Missouri are at risk for exposure to B. lonestari after A. americanum tick bite, a possible cause of erythema migrans-like rash illness in this region. Derivation of the complete coding sequence for B. lonestari flaB is also reported.

Erythematous rashes that are suggestive of early Lyme disease have been associated with the bite of Amblyomma americanum ticks, particularly in the southern United States. However, Borrelia burgdorferi, the causative agent of Lyme disease, has not been cultured from skin biopsy specimens from these patients, and diagnostic serum antibodies usually have not been found. Borrelia lonestari sp nov, an uncultured spirochete, has been detected in A. americanum ticks by DNA amplification techniques, but its role in human illness is unknown. We observed erythema migrans in a patient with an attached A. americanum tick. DNA amplification of the flagellin gene flaB produced B. lonestari sequences from the skin of the patient that were identical to those found in the attached tick. B. lonestari is a probable cause of erythema migrans in humans.

VlsE, the variable surface antigen of Borrelia burgdorferi , contains an immunodominant conserved region named IR 6 . In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C 6 ) with the IR 6 sequence was explored. Sensitivity was assessed with serum samples ( n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C 6 peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.

In Colorado, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease, is maintained in an enzootic cycle between Ixodes spinipalpis ticks and Neotoma mexicana rats (27). The frequencies of flagellin (fla), 66-kDa protein (p66), and outer surface protein A (ospA) alleles were examined in 71 B. burgdorferi isolates from samples from Colorado. Approximately two-thirds of these samples were isolates from I. spinipalpis ticks that had been cultured in BSK-H medium prior to DNA extraction. The remaining samples were from total DNA extracted directly from infected I. spinipalpis ticks. A portion of each gene was amplified by PCR and screened for genetic variability by single-strand conformation polymorphism (SSCP) analysis. We identified three alleles in the fla gene, seven in the p66 gene, and seven in the ospA gene. Sequencing verified that the amplified products originated from B. burgdorferi template DNA and indicated 100% sensitivity and specificity of the SSCP analysis. The frequencies of the p66 and ospA alleles were significantly different between cultured and uncultured spirochetes. The number of three-locus genotypes and the genetic diversity of alleles at all loci were consistently lower in cultured spirochetes, suggesting that culturing of B. burgdorferi in BSK-H medium may select for specific genotypes.

Outbred mice were immunized with the soluble fraction of a crude Escherichia coli lysate containing either recombinant outer surface protein C (OspC or P39 of Borrelia burgdorferi B31 (low passage). Following seroconversion, the mice were challenged with an infectious dose of B. burgdorferi B31 via the natural transmission mode of tick bite. Three mice immunized with P39 were not protected; however, all 12 of the recombinant OspC-immunized mice were protected from infection as assayed by culture and serology. Although OspC has been shown to be a protective immunogen against challenge with in vitro-cultured borrelia administered by needle, this study is the first to demonstrate OspC effectiveness against tick-borne spirochetes. Following feeding, all ticks still harbored B. burgdorferi, suggesting that the mechanism of protection is not linked to destruction of the infectious spirochete within the tick. In a separate experiment, groups of four mice were immunized with protein fractions from B. burgdorferi B31 purified by preparative gel electrophoresis in an attempt to identify potential protective antigens. Many of these mice developed high-titer-antibody responses against OspC, but curiously the mice were susceptible to B. burgdorferi infection via tick bite. These results suggest that the protective epitope(s) on OspC is heat sensitive/conformational, a finding which has implications in vaccine development.

Efforts to develop a recombinant vaccine for Lyme disease have focused on using the outer surface protein A (OspA) of Borrelia burgdorferi as an immunogen. We evaluated the effectiveness of an unlipidated recombinant OspA as a vaccine in hamsters. This molecule is soluble and can be produced in high yield in Escherichia coli, characteristics that permit simple and relatively low cost production. Vaccination with unlipidated OspA protected a substantial portion of animals--59-79%, depending on the challenge strain and route--against moderate doses of spirochetes delivered either by injection or by bite of infected nymphal ticks (Ixodes scapularis). The instances of vaccine failure were associated with development of low levels of antibody to a particular OspA epitope, one defined by mAb LA-2. At least 50 ng ml-1 of LA-2 equivalent antibody was necessary for protection of hamsters. Lower LA-2 equivalent antibody concentrations occurred in unprotected animals in the presence of high-titered polyclonal antibody to native OspA. A competitive binding assay to quantitate this serum fraction is described that should be of use in monitoring the quality of the antibody response to OspA in vaccine trials. Concentrations of LA-2 equivalent antibody parallel the ability of the serum specimens to inhibit the growth of B. burgdorferi in culture.

Valuable insights into the pathogenesis and immunoprophylaxis of Lyme disease are beginning to emerge from studies in animal models. This review highlights two animal models: the mouse, which has allowed us to investigate the role of both the immune response and spirochete phenotype in determining the outcome of the disease; and the Rhesus monkey, which manifests signs of nerve involvement, in addition to showing erythema migrans and arthritis.

An intensive enzootic cycle of Borrelia burgdorferi was seen in populations of the Mexican wood rat, Neotoma mexicana, and Ixodes spinipalpis ticks in northern Colorado. Cultures of rodent ear tissue and ticks yielded 63 spirochetal isolates: 38 N. mexicana, 2 Peromyscus difficilis, and 23 I. spinipalpis. All 63 isolates were identified as B. burgdorferi sensu lato by polymerase chain reaction; a representative subset was characterized as B. burgdorferi by SDS-PAGE and immunoblotting. A tick-derived spirochete isolate was infectious to laboratory mice and I. scapularis, the principal vector of Lyme disease in endemic areas of the United States. The risk of human contact with infected I. spinipalpis appears to be minimal from this epidemiologically silent focus in northern Colorado, since this tick is restricted to wood rat nests in this semiarid environment.

Attenuated mutants of Venezuelan equine encephalitis virus (VEE) were isolated by selection for rapid penetration of cultured cells (R. E. Johnston and J. F. Smith, 1988, Virology 162, 437-443). Sequence analysis of these mutants identified candidate attenuating mutations at four loci in the VEE E2 glycoprotein gene: a double mutation at E2 codons 3 and 4, and single substitutions at E2 76, 120, and 209. Each candidate mutation was reproduced in an isogenic recombinant VEE strain using site-directed mutagenesis of a full-length cDNA clone of VEE. Characterization of these molecularly cloned mutant viruses showed that mutation at each of the four loci in the E2 gene was sufficient to confer both the accelerated penetration and attenuation phenotypes. Inoculation of the molecularly cloned viruses into rodent models that differ in their response to VEE suggested that individual mutations affected different aspects of VEE pathogenesis. Full-length clones containing multiple mutations were produced by combining independently attenuating mutations. Molecularly cloned viruses carrying two or three mutations were more attenuated in sensitive animal models than viruses which contained any single mutation alone. However, these highly attenuated strains still retained the ability to induce an immune response sufficient to protect against a high dose challenge with virulent VEE. These results indicate that production of a molecularly cloned live virus vaccine for VEE is feasible.

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.

In this report we describe conditions for polyclonal activation of small numbers of highly purified mouse B lymphocytes. Three signals are required for induction of DNA synthesis by the particular subset of small B lymphocytes investigated: a signal delivered by antibodies specific for the IgM receptor expressed on the B cell membrane; a signal delivered by a T cell-derived factor (B cell growth factor [BCGF]); and a signal delivered by the macrophage-derived factor interleukin 1 (IL-1). The conclusion that IL-1 has B cell co-stimulator activity is based on the findings that highly purified preparations of mouse and human IL-1 have the capacity to cause proliferation in B cells treated with anti-IgM and BCGF. Such cultures show an absolute dependence on exogenously added IL-1 when 2-mercaptoethanol is omitted from the medium. BCGF and IL-1 each act in a non-antigen-specific, non-H-2-restricted, synergistic manner. Their requirement is not observed when B cells are cultured at high density, presumably reflecting accessory cell contamination and endogenous factor production under these conditions. The B cell activation induced by these three signals is restricted to proliferation without the production of antibody-forming cells.

Nucleotide sequence analysis of cDNA clones covering the entire genomes of Trinidad donkey (TRD) Venezuelan equine encephalitis (VEE) virus and its vaccine derivative, TC-83, has revealed 11 differences between the genomes of TC-83 virus and its parent. One nucleotide substitution and a single nucleotide deletion occurred in the 5′- and 3′- noncoding regions of the TC-83 genome, respectively. The deduced amino acid sequences of the nonstructural polypeptides of the two viruses differed only in a conservative Ser (TRD) to Thr (TC-83) substitution in nonstructural protein (nsP) three at amino acid position 260. The two silent mutations (one each in El and E2), one amino acid substitution in the El glycoprotein, and five substitutions in the E2 envelope glycoprotein of TC-83 virus were reported previously ( B. J. B. Johnson, R. M. Kinney, C. L. Kost, and D. W. Trent, 1986 , J. Gen. Viral . 67, 1951–1960). The genome of TRD virus was 11,444 nucleotides long with a 5′-noncoding region of 44 nucleotides. The carboxyl terminal portion of VEE nsP3 contained two peptide segments (7 and 34 amino acids long) that were repeated with high fidelity. The open reading frame of the nonstructural polyprotein was interrupted by an in-frame opal termination codon between nsP3 and nsP4, as has been reported for Sindbis, Ross River, and Middelburg viruses. The deduced amino acid sequences of the VEE TRD nsP1, nsP2, nsP3, and nsP4 polypeptides showed 60–66%, 57–58%, 35–44%, and 73-71% identity with the aligned sequences of the cognate polypeptides of Sindbis and Semliki Forest viruses, respectively. The lack of homology in the nsP3 of the viruses is due to sequence variation in the carboxyl terminal half of this polypeptide.

We report here a factor (B cell growth factor) found in induced supernatants of the mouse thymoma EL4 that co-stimulates with anti-IgM antibodies in short-term cultures of purified B lymphocytes to induce polyclonal B cell proliferation but not antibody-forming cell production. The factor is not mitogenic for resting B cells and interacts with anti-IgM-activated B cells in a non-H-2-restricted manner. Absorption studies and molecular weight analysis reveal the factor is distinct from interleukin 2. This factor synergises with antigen, interleukin 2, and an interleukin 2-free, B cell growth factor-free T cell supernatant that contains T cell-replacing factor to produce erythrocyte-specific plaque-forming cells in cultures of highly purified B cells.

Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.

Human immunodeficiency virus (HIV) was detected in bedbugs (Cimex hemipterus) up to 8 d after oral exposure to highly concentrated virus in blood meals, but no virus replication was observed. HIV did not replicate in either intraabdominally inoculated bedbugs or intrathoracically inoculated mosquitoes (Toxorhynchites amboinensis). The virus was not detected in bedbug feces. Mechanical transmission of HIV by bedbugs could not be demonstrated in an in vitro model. The persistence of HIV in an insect or on its mouthparts is one of many factors necessary for mechanical transmission in nature. The risk of insect transmission of HIV appears to be extremely low or nonexistent.

The nucleotide and deduced amino acid sequences of the structural proteins of the TC-83 vaccine strain of Venezuelan equine encephalitis (VEE) virus have been determined from a cDNA clone containing the 26S mRNA coding region. A cDNA clone encoding the equivalent region of the virulent parent VEE virus [Trinidad donkey strain (TRD)] has been sequenced previously. Comparison of the sequences of the TC-83 and TRD cDNA clones revealed 13 nucleotide differences. Neither the organization of the structural proteins (5'-capsid-E3-E2-6K-E1-3') nor the length (3762 nucleotides) of the open reading frame coding for the viral polyprotein precursor was altered during attenuation. Of the 13 nucleotide differences between the cDNA clones of TC-83 and TRD, nine occurred in the dominant population of the respective genomic RNAs from plaque-purified viruses. Six of the nine mutations were clustered in the E2 surface glycoprotein gene. All five of the nucleotide changes which produced non-conservative amino acid substitutions in the encoded proteins were located in the E2 gene. Two mutations occurred in the E1 glycoprotein gene; one was silent and the other did not alter the chemical character of the E1 protein. One nucleotide difference was found in the non-coding region immediately preceding the 5'-end of the 26S mRNA. The E2 and non-coding region mutations are candidates for the molecular determinants of VEE virus neurovirulence.