DNA sequence and analysis of human chromosome 9.

Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6-8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.

Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG).

Deletion of the long arm of chromosome 20 represents the most common chromosomal abnormality associated with the myeloproliferative disorders (MPDs) and is also found in other myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Previous studies have identified a common deleted region (CDR) spanning approximately 8 Mb. We have now used G-banding, FISH or microsatellite PCR to analyse 113 patients with a 20q deletion associated with a myeloid malignancy. Our results define a new MPD CDR of 2.7 Mb, an MDS/AML CDR of 2.6 Mb and a combined 'myeloid' CDR of 1.7 Mb. We have also constructed the most detailed physical map of this region to date--a bacterial clone map spanning 5 Mb of the chromosome which contains 456 bacterial clones and 202 DNA markers. Fifty-one expressed sequences were localized within this contig of which 37 lie within the MPD CDR and 20 within the MDS/AML CDR. Of the 16 expressed sequences (six genes and 10 unique ESTs) within the 'myeloid' CDR, five were expressed in both normal bone marrow and purified CD34 positive cells. These data identify a set of genes which are both positional and expression candidates for the target gene(s) on 20q.

Flavivirus DNA vaccines: current status and potential.

The use of DNA-based vaccines is a novel and promising immunization approach for the development of flavivirus vaccines. This approach has been attempted in vaccine development for various virus species, including St. Louis encephalitis, Russian spring-summer encephalitis, Central European encephalitis, dengue serotypes 1 and 2, Murray Valley encephalitis, Japanese encephalitis, and West Nile viruses. However, very little is known about the factors affecting its efficacy. Recently, we demonstrated that a single intramuscular immunization of DNA vaccine of Japanese encephalitis and West Nile viruses protected mice and horses from virus challenge. Administration of these recombinant plasmid vectors resulted in endogenous expression and secretion of extracellular virus-like particles that correlated well with the induction of protective immunity. These results provided evidence that the virus-like particles composed of premembrane/membrane and envelope proteins are essential for eliciting immune responses similar to those induced by live, attenuated virus vaccines. The biosynthesis and protein processing of premembrane/membrane and envelope proteins that preserve the native conformation and glycosylation profiles identical to virion proteins could be determined by the effectiveness of the transmembrane signal sequence located at the amino-terminus of premembrane protein. The use of DNA vaccines in multivalent and/or combination vaccines designed to immunize against multiple flaviviruses is also a promising area of development.

Antibody prophylaxis and therapy for flavivirus encephalitis infections.

The outbreak of West Nile (WN) encephalitis in the United States has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. Although equine WN vaccines are currently being developed, a WN vaccine for humans is years away. There is also no specific therapeutic agent for flaviviral infections. The incidence of human WN virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the vaccine's economic feasibility. It has been shown, however, that prophylactic application of antiflaviviral antibody can protect mice from subsequent virus challenge. This model of antibody prophylaxis using murine monoclonal antibodies (MAbs) has been used to determine the timing of antibody application and specificity of applied antibody necessary for successful prophylaxis. The major flaviviral antigen is the envelope (E) glycoprotein that binds cellular receptors, mediates cell membrane fusion, and contains an array of epitopes that elicit virus-neutralizing and nonneutralizing antibodies. The protective efficacy of an E-glycoprotein-specific MAb is directly related to its ability to neutralize virus infectivity. The window for successful application of prophylactic antibody to prevent flaviviral encephalitis closes at about 4 to 6 days postinfection concomitant with viral invasion of the brain. Using murine MAbs to modify human disease results in a human antimouse antibody (HAMA) response that eventually limits the effectiveness of subsequent murine antibody applications. To reduce the HAMA response and make these MAbs more generally useful for humans, murine MAbs can be "humanized" or human MAbs with analogous reactivities can be developed. Antiflaviviral human or humanized MAbs might be practical and cost-effective reagents for preventing or modifying flaviviral diseases.

Relationships of bunyamwera group viruses by neutralization.

A standardized serum dilution plaque reduction neutralization test was used for cross-neutralization studies of 23 strains of Bunyamwera serogroup viruses. Antigenic relationships were determined by inspection of the neutralization tests results as well as by numerical taxonomic analysis. Based on these analyses five complexes, containing 1-11 viruses, were distinguished. Little or no cross-reactivity was observed between viruses of different complexes. Three of the viruses tested were indistinguishable from prototypes and probably represent strains or varieties of those prototypes. A tentative classification scheme for the Bunyamwera group is presented.

Identification of Highlands J virus from a Florida horse.

A virus, strain 64A-1519, isolated from the brain of a horse dying of encephalitis in Florida in 1964, was identified as western equine encephalomyelitis (WEE) virus. Recently, we used polyclonal and monoclonal immune reagents to identify this isolate by comparing it to 2 strains of WEE virus and to Highlands J (HJ) virus in hemagglutination-inhibition, immunofluorescent antibody, and plaque-reduction neutralization tests. These tests demonstrate that strain 64A-1519 is a strain of HJ virus distinct from WEE virus.

Arbovirus investigations in Argentina, 1977-1980. III. Identification and characterization of viruses isolated, including new subtypes of western and Venezuelan equine encephalitis viruses and four new bunyaviruses (Las Maloyas, Resistencia, Barranqueras, and Antequera).

Forty viruses isolated from mosquitoes between 1977 and 1980 in Argentina have been identified and characterized. Nineteen strains of VEE virus, identical by neutralization (N) tests, were shown by hemagglutination-inhibition tests with anti-E2 glycoprotein sera to represent a new subtype VI of the VEE complex. RNA oligonucleotide fingerprints of this virus were distinct from subtype I viruses. The virus was not lethal for English short-haired guinea pigs, indicating that it is probably not equine-virulent. Three strains of a member of the WEE virus complex were shown to differ by N tests in 1 direction from prototype WEE virus. The new WEE subtype was also found to be distinct by RNA oligonucleotide mapping. Its vector relationships indicate that it is an enzootic virus, and it has not been associated with equine disease. A new member of the Anopheles A serogroup was identified, shown to be most closely related to Lukuni and Col An 57389 viruses, and given the name Las Maloyas virus. A strain of Para virus (Bunyaviridae, Bunyavirus) was identified. Six isolates, representing 3 new viruses morphologically resembling bunyaviruses are described; the names Antequera, Barranqueras, and Resistencia are proposed for these agents, which were all isolated from Culex (Melanoconion) delpontei in Chaco Province. No serologic relationships between these viruses and other bunyaviruses were found. Since they are antigenically interrelated, they form a new (Antequera) serogroup. Eight Gamboa serogroup viruses and 2 strains of St. Louis encephalitis virus were also identified.

Identification of monoclonal antibodies capable of differentiating antigenic varieties of eastern equine encephalitis viruses.

We have isolated and characterized 3 monoclonal antibody (Mab) reagents useful in the serological identification of varieties of eastern equine encephalitis (EEE) viruses. These antibodies were specific for the E1 glycoprotein of their homologous viruses. One Mab, 1B5C-3, reacted specifically with all North American (NA) EEE viruses isolated over a 50 year period. This antigenic stability of NA isolates was genetically confirmed by oligonucleotide fingerprinting. Evolutionary stability is a unique feature among alphaviruses. The Mab, 1C1J-4 reacted specifically with 1 South American isolate of EEE virus. A third Mab, 1B1C-4, was EEE virus complex reactive. While none of these antibodies had virus neutralizing activity, the identified reactivities could be demonstrated in the more rapid serological tests of enzyme-linked immunosorbent assay and indirect immunofluorescence.

A recombinant particulate antigen of Japanese encephalitis virus produced in stably-transformed cells is an effective noninfectious antigen and subunit immunogen.

A COS-1 cell line, stably transformed by a plasmid encoding the premembrane and envelope glycoproteins of Japanese encephalitis virus, produced a noninfectious recombinant antigen expressed as extracellular particles. Extracellular particles purified by equilibrium density centrifugation in sucrose gradients followed by electron microscopy were characterized as spherical particles with an average diameter of approximately 30 nm and a buoyant density of 1.15 g/cc. Purified extracellular particles were shown by western blot to contain premembrane, membrane and envelope proteins. The gradient-purified particles exhibited hemagglutination activity at the same pH optimum (6.6) as Japanese encephalitis virus. Recombinant antigen from cell culture fluid was concentrated by precipitation with polyethylene glycol and evaluated for immunogenicity in 8-10-week-old ICR mice. Groups of five mice received only one immunization of recombinant antigen with or without Freund's incomplete adjuvant. Mice immunized with recombinant antigen plus Freund's incomplete adjuvant elicited the highest anti-viral titers as determined by both enzyme-linked immunosorbent assay (ELISA) and plaque-reduction neutralization tests. The polyethylene glycol-concentrated recombinant antigen was also evaluated for use in IgM antibody-capture ELISA and indirect IgG ELISA. The IgM-capture ELISA results using recombinant antigen correlated well with the results of a similar test using Japanese encephalitis virus-infected mouse brain antigen for the analysis of serum samples from patients with symptoms of acute encephalitis. Similar IgG titers were observed in an indirect ELISA comparing recombinant antigen and purified Japanese encephalitis virus as plate-bound antigens. Based on these studies, this entirely safe, easily produced antigen that expresses authentic Japanese encephalitis virus envelope glycoprotein would provide an excellent alternative to standard viral antigens used in various ELISA formats.

Factors associated with prognosis for survival and athletic use in foals with septic arthritis: 93 cases (1987-1994).

OBJECTIVE: To identify factors affecting the prognosis for survival and athletic use in foals with septic arthritis. DESIGN: Retrospective study. ANIMALS: 93 foals with septic arthritis. PROCEDURE: Medical records were reviewed to obtain clinical findings, laboratory test results, radiographic findings, treatment method, and outcome. Race records for Thoroughbreds and Standardbreds were evaluated to determine whether foals subsequently raced and whether they raced successfully. RESULTS: 43 foals had 1 affected joint, 44 foals had multiple affected joints, and number of affected joints was not recorded for 6 foals. The femoropatellar and tarsocrural joints were most commonly affected. Osteomyelitis or degenerative joint disease were detected in 59% (46/78) of foals. Failure of passive transfer, pneumonia, and enteritis were common. Foals were treated with lavage, lavage and intra-articular administration of antibiotics, lavage and arthroscopic debridement with or without partial synovectomy, or lavage and arthrotomy to debride infected bone and systemic administration of antibiotics. Seventy-three foals survived to be discharged from hospital, and approximately a third raced. Isolation of Salmonella spp from synovial fluid was associated with an unfavorable prognosis for survival and multisystem disease was associated with an unfavorable prognosis for survival and ability to race; other variables were not significantly associated with survival and ability to race. CONCLUSIONS AND CLINICAL RELEVANCE: With treatment, the prognosis for survival of foals with septic arthritis was favorable, whereas prognosis for ability to race was unfavorable. Multisystem disease, isolation of Salmonella spp from synovial fluid, involvement of multiple joints, and synovial fluid neutrophil count > or = 95% at admission may be of prognostic value.

Biochemical and biological characteristics of epitopes on the E1 glycoprotein of western equine encephalitis virus.

Antigenic determinants identified by monoclonal antibodies (Mabs) on the E1 glycoprotein of western equine encephalitis (WEE) virus have been characterized by their serological activity, requirements for secondary structure, expression on the mature virion, and their role in protecting animals from WEE virus challenge. On the basis of a cross-reactivity enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay, eight antigenic determinants (epitopes) on the E1 glycoprotein have been identified, ranging in reactivity from WEE-specific to alphavirus group reactive. No neutralization of virus infectivity was demonstrable with any of the Mabs. An alphavirus group-reactive hemagglutination (HA) site, a WEE complex-reactive HA site, and a WEE virus-specific HA site were identified. Spatial arrangement of these epitopes was determined by a competitive binding ELISA. Four competition groups defining three distinct antigenic domains were identified. Antibodies directed against four E1 epitopes were capable of precipitating the E1/E2 heterodimer from infected cells or purified virus disrupted with nonionic detergents. These same antibodies precipitated only E1 in the presence of 0.1% SDS. That E1 conformation was important was shown by the inability of antibodies specific for seven of the epitopes to bind to virus denatured in 0.5% SDS. As determined by equilibrium gradient analysis of virus-antibody mixtures, four epitopes were found to be fully accessible on the mature virion, three epitopes were inaccessible, and one epitope was partially accessible to antibody binding. Antibodies specific for three epitopes were able to passively protect mice from WEE virus challenge.

Localization of a protective epitope on a Venezuelan equine encephalomyelitis (VEE) virus peptide that protects mice from both epizootic and enzootic VEE virus challenge and is immunogenic in horses.

In order to define more precisely the protective epitope encoded within the first 25 amino acids (aa) of the E2 glycoprotein of the Trinidad donkey strain of Venezuelan equine encephalomyelitis (VEE) virus, we examined the immunogenicity of smaller peptides within the first 19 aa. pep1-9 and pep3-10 elicited virus-reactive antibody, but failed to protect mice from virus challenge. Additionally, pep3-10 was identified by a competitive binding assay using overlapping peptide octamers as the putative binding site of the antipeptide monoclonal antibody (mAb) 1A2B-10. Since the E2 amino-terminal sequence for all VEE subtype viruses is conserved, we tested the protective capacity in mice of passively transferred mAb 1A2B-10 and found it to protect from both epizootic and enzootic VEE virus challenge. Since horses are an important natural host for VEE virus, pep1-19 was used to immunize horses and was found to be immunogenic and to elicit virus-reactive antibody.

Synthetic peptides of Venezuelan equine encephalomyelitis virus E2 glycoprotein. I. Immunogenic analysis and identification of a protective peptide.

Fourteen peptides representing 67% of the extramembranal domain of the Venezuelan equine encephalomyelititis (VEE) virus E2 glycoprotein were synthesized and analyzed to determine their antigenic, immunogenic, and protective capacities. Thirteen of 14 peptides elicited antibody for the homologous peptide. Thirteen peptides elicited antiviral antibody that recognized either the Trinidad (TRD) strain of VEE virus or the TC-83 vaccine derivative, or both. Two peptides, VE2pep01(TC-83) and VE2pep01(TRD), protected significant numbers of mice from TRD virus challenge. The majority of the peptides were reactive with antisera from mice immunized with the various subtypes of VEE virus. A competition assay using antipeptide antibodies to block virus binding of anti-VEE virus monoclonal antibodies corroborated previous studies on the spatial relationship of E2 epitopes and provided evidence for a spatial overlap of the E2 amino terminus with a domain composed of residues 180-210.

Synthetic peptides of Venezuelan equine encephalomyelitis virus E2 glycoprotein. III. Identification of a protective peptide derived from the carboxy-terminal extramembranal one-third of the protein.

To complete our analysis of the E2 glycoprotein of Venezuelan equine encephalomyelitis (VEE) virus, we prepared six synthetic peptides corresponding to the extramembranal carboxy-terminal one-third of the protein. NIH-Swiss mice were immunized with the peptides, and antipeptide and antiviral titers were determined by enzyme-linked immunosorbent assay (ELISA). Challenge studies revealed that peptide 13 (amino acids 241-265) protected 60-70% of virus-challenged mice. Although the other peptides generally elicited antipeptide ELISA titers but no or low antiviral titers and did not protect mice, significant E2 reactivity was found in immunoblots. These results provide the first direct evidence that much of the E2 carboxy-terminal domain is cryptic in the VEE virion, even when virus was bound to polystyrene ELISA plates.

A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice.

Plasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50, 000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.

Antibodies to arboviruses in an Alaskan population at occupational risk of infection.

A total of 435 United States Geological Survey and United States Forest Service workers in Alaska were studied for serologic evidence of past infections with four arboviruses known or suspected to be human pathogens. Of the personnel tested, 36 (8.3%) had the neutralizing antibody to Jamestown Canyon but not snowshoe hare virus, 6 (1.4%) had the antibody to snowshoe hare but not Jamestown Canyon virus, 53 (12.2%) had the antibody to both viruses, 17 (3.9%) had the antibody to Northway virus, and 15 (3.4%) had the antibody to Klamath virus. The indices most significantly correlated with presence of the Jamestown Canyon and snowshoe hare antibodies were the amount of fieldwork (p less than 0.001 for both antibodies) and the duration of employment by the agencies (p less than 0.0001 for Jamestown Canyon and 0.004 for snowshoe hare). The antibody to the four arboviruses also correlated strongly with a history of travel in certain remote or wilderness areas in Alaska (p values ranged from less than 0.001 to 0.086).

Synthetic peptides derived from the deduced amino acid sequence of the E-glycoprotein of Murray Valley encephalitis virus elicit antiviral antibody.

We used computer analysis to study hydrophilicity, homology, surface accessibility, molecular flexibility, and secondary structure of the deduced amino acid sequence of the flavivirus envelope (E)-glycoprotein. Using the results, we modified the E-glycoprotein antigenic structure proposed by Nowak and Wengler (1987, Virology, 156, 127-137). Our model predicts considerable overlaps in the previously defined domains. We have prepared 11 synthetic peptides from the deduced amino acid sequence of the E-glycoprotein of Murray Valley encephalitis (MVE) virus and analyzed their immunogenicity. Peptides derived from the redefined R1 and R2 domains elicit antiviral antibody. Nine of these peptides are recognized by polyclonal antiviral antibodies; however, none are consistently recognized by monoclonal antibodies. Peptides derived from the R1 domain demonstrate MVE virus specificity, and 1 peptide elicited low-level virus neutralizing antibody. Spatial overlap of the domains was defined by competitive binding assays between antipeptide antisera and radioactive monoclonal antibodies. These results indicate that synthetic peptides aid in defining flavivirus antigenic structure, and may serve as possible type-specific diagnostic reagents.

Antigenic structure of the Murray Valley encephalitis virus E glycoprotein.

To complement our battery of St Louis encephalitis (SLE) virus monoclonal antibodies (MAbs), we isolated and characterized MAbs reactive with another member of the SLE virus serocomplex, Murray Valley encephalitis (MVE) virus. From 40 fusion products, we isolated 10 stable hybridomas. The combination of SLE and MVE virus MAbs defined eight epitopes on the MVE envelope (E) glycoprotein. Six of these epitopes (E-1a, E-1c, E-1d, E-3, E-4a, E-4b) were identical to those previously demonstrated on SLE virus. Two new epitopes (E-5 and E-6) were also identified. As with SLE virus, the MVE E-1c epitope elicited the most potent virus-neutralizing and protective MAb. Unlike SLE virus, however, one of the cross-reactive epitopes (E-5) elicited neutralizing antibody and protected animals from MVE virus challenge. These results indicate that, while the antigenic domains on viruses within the SLE virus serocomplex are quite similar, epitopes involved in virus neutralization or protection from virus challenge may vary and can be topologically distinct.

In vitro mechanisms of monoclonal antibody neutralization of alphaviruses.

We have previously identified at least eight epitopes on the E2 glycoprotein of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83 by using monoclonal antibodies (MAbs). Several of these antibodies identified a critical neutralization (N) domain in competitive binding assays. Passive transfer of these MAbs protected animals from a lethal virus challenge. Using radioactive, purified virus as a marker, we have demonstrated that antibody-mediated virus N, preattachment, can be effected by one of three mechanisms. Interaction of antibody can block virus attachment to susceptible Vero or human embryonic lung cells. The MAbs that were most efficient at blocking attachment were those that defined epitopes spatially proximal to the E2c epitope. The E2c MAbs were, however, the most efficient antibodies for neutralizing virus postattachment. Other E2 MAbs were unable to efficiently block virus attachment to cells; however, resulting replication as monitored by plaque assay or intracellular viral RNA synthesis could not be detected. One novel MAb that defined the E2f epitope appeared to enhance virus attachment to Vero cells, but not BHK-21 or LLC-MK2 cells, by stabilizing virus-cell interaction. This antibody did, however, efficiently neutralize virus infectivity. Once virus had attached to cells, the ability of most MAbs to neutralize infectivity was diminished, except for E2c MAbs. On a molar basis antibody Fab fragments were less efficient than intact antibody at blocking virus attachment.

Use of a new synthetic-peptide-derived monoclonal antibody to differentiate between vaccine and wild-type Venezuelan equine encephalomyelitis viruses.

We have prepared a murine monoclonal antibody (MAb) capable of distinguishing between wild-type Venezuelan equine encephalomyelitis (VEE) virus and the TC-83 vaccine derivative. This MAb, 1A2B-10, was derived from immunization with a synthetic peptide corresponding to the first 19 amino acids of the E2 glycoprotein of Trinidad donkey VEE virus. The MAb reacts with prototype viruses from all naturally occurring VEE subtypes except subtype 6 in an enzyme-linked immunosorbent assay. It does not react with TC-83 virus or members of the western and eastern equine encephalitis virus complex or with Semliki Forest virus. This antibody will also differentiate between TC-83 and Trinidad donkey VEE virus in indirect immunofluorescence assays with virus-infected Vero cells.