A water-soluble derivative of prazosin prazosinamine hydrochloride [1-(4'-amino-6',7'-dimethoxyquinazolin-2'-yl)-4-(6''-aminohexanoyl) piperazine hydrochloride], reversibly inhibits the calcium-mobilizing action of alpha 1-adrenergic agonists in the perfused rat liver.

A newly-synthesized derivative of prazosin, prazosinamine hydrochloride, was examined for its ability to antagonize the interaction of the alpha 1-adrenergic agonist phenylephrine with liver cells. Using a Ca2--selective electrode to measure changes in perfusate Ca2+ concentration, prazosinamine was found to be as effective as prazosin in inhibiting the phenylephrine-induced efflux of Ca2+ from the perfused liver. Maximal and half-maximal inhibition occurred at 150 nM and 25 nM prazosinamine, respectively. Prazosinamine appears to share the alpha 1-specificity of prazosin, but has other unique and desirable properties. Its solubility in aqueous media is about three orders of magnitude higher than that of prazosin. Also, its antagonistic effects are rapid in onset, and are reversed within seconds of terminating its infusion into the liver. These attributes seem to make this agent more useful than prazosin for adrenergic receptor studies in perfused tissues. The molecule can also be readily coupled to other ligands.

Immune response to a Murray Valley encephalitis virus epitope expressed in the flagellin of an attenuated strain of Salmonella.

Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as carriers of foreign antigens. These recombinant strains can elicit a heterologous immune response when injected into animals, demonstrating potential for their use in the construction of many vaccines. In the present study, a B-cell epitope of Murray Valley encephalitis virus (MVE) was identified and expressed in a Salmonella strain to evaluate its potential to induce a specific immune response to MVE. A synthetic oligonucleotide encoding the B-cell epitope (residues E201-224) of the envelope protein of MVE was inserted into the cloned flagellin gene of the Salmonella strain. The construct was sequenced to ensure correct orientation of the epitope. Expression of the epitope was demonstrated by Western blot analysis and immunogold electron microscopy with monoclonal antibody specific to the epitope. Electron microscopy analysis revealed multiple copies of the epitope along the flagella. The recombinant Salmonella carrying the hybrid flagellin gene elicited an immune response to the MVE epitope in a mouse model. The MVE-specific antibodies partially neutralised the virus in vitro. The significance of this system for engineering vaccines for other medically important flaviviruses is discussed.

Direct sequence analysis of amplified dengue virus genomic RNA from cultured cells, mosquitoes and mouse brain.

A method is described for direct sequence analysis of selected regions of dengue virus genomic RNA in infected tissues. Using specific primers, total high-molecular-weight infected-cell RNA is reverse transcribed to single-stranded (ss) complementary DNA, amplified using the polymerase chain reaction (PCR) and sequenced using ssDNA obtained after lambda exonuclease digestion of one strand of the PCR product (R.G. Higuchi and H. Ochman, Nucleic Acids Research, 17, 5865, 1989). Sequence data for the envelope protein gene of two dengue-3 virus isolates were obtained using RNA from small numbers (10(5)) of cultured mosquito or monkey kidney cells, from one mg of infected mouse brain and from 1/300th of an infected Toxorhynchites amboinensis mosquito. Independent determinations showed that errors occurring during reverse transcription or PCR were not represented to a significant degree in the sequence of the amplified DNA. The method does not depend on extensive passaging of virus or large-scale growth to generate material for sequencing and therefore provides a means of obtaining sequence data for unadapted dengue virus isolates.

Post-swim orthostatic intolerance in a marathon swimmer.

Two swims (1993 and 1994) are described which led to post-swim orthostatic intolerance and one episode of syncope in a 50/51-yr-old well-trained and experienced marathon swimmer. The swims of 33 km and 38 km took 12 h 30 s and 17 h 35 min, respectively. Water temperature in each swim was above 23 degrees C and rectal core-temperature stayed above 37.0 degrees C. Air temperatures differed, ranging from 23 degrees to 37 degrees C and 15 degrees to 21 degrees C, respectively. Regular fluid consumption totalled approximately 5.0 and 6.0 1, respectively. Fifteen minutes after completing the 1993 swim, the swimmer experienced orthostatic intolerance and fainted at the lakeside; hospital tests revealed an elevated creatine phosphokinase (CK) of 521 U.l-1. The 1994 swim was abandoned due to severe muscle cramps and CK was found to be markedly elevated at 909 U.l-1. Orthostatic intolerance was recorded in both cases; however, no cardiac abnormalities were found. After overnight rest and intravenous saline infusions of 3.0 and 1.5 l, respectively, the orthostatic intolerance was relieved. Based on previous descriptions of exercise-associated collapse in marathon runners, the swimmer's orthostatic intolerance and syncope are attributed to blood pooling in his legs due to inactivation of the venous muscle pump on completion of the swim.

Comparison of the structure of the immunoglobulins from horse serum.

A study of the chemical structure of the horse immunoglobulins IgG and IgA(T) has shown that the amino acid contents of the peptide chains are very similar. These globulins differ most markedly in the products of papain digestion. IgG gives 3.5s products, whereas IgA(T) gives a 5s fraction and smaller components. This difference appears to be associated with the presence of an additional easily reducible disulphide bond in the Fd fragment of the heavy chain. There is two to three times as much carbohydrate in IgA(T) as in IgG. In both, this is in the heavy chain and in IgA(T) more than half is covalently bound to the Fd fragment. The differences in antigenic specificity between horse IgG and IgA(T) appear to be due to structural differences in the Fc fragment.

The antigen-binding capacity of the peptide chains of horse antibodies.

The antigen-binding capacity of the peptide chains of horse anti-(diphtheria toxin) has been studied by using (125)I-labelled toxoid and electrophoresis of antibody-antigen mixtures on cellulose acetate. The heavy chains retained about 20% of the activity of the whole antibody and the light chains less than 5%. Recombination of specific heavy and light chains gave about 60% recovery of activity and recombination of specific heavy chains and non-specific light chains about 40% recovery. It is suggested that these results favour the heavy chain as the major site of the antigen-binding activity.

Changes in the dengue virus major envelope protein on passaging and their localization on the three-dimensional structure of the protein.

To help define the molecular events involved in dengue virus adaptation during serial passage in vivo and in cultured cells, we have sequenced the structural protein genes of three dengue type 3 isolates after intracerebral passage in mice and after passage in cultured monkey kidney (Vero) and Aedes albopictus (mosquito) cells. Passaging in each host selected for amino acid changes in the envelope protein E and occasionally in prM but not in the capsid protein. Most changes were first apparent within five passages. Nineteen of twenty mutations in the structural protein genes resulted in amino acid changes concentrated on 12 residues; 9 of the 12 amino acid changes were at residues which are conserved between the four dengue virus serotypes. Certain amino acid changes were repeatedly selected on passage in cell culture. In six independent Vero cell passage series, changes were observed in E at residues 191 (four times), 202 (twice), 266 and 268 (three times), and 291; change in prM was seen in two passage series at residue 26. Two independent passage series in mosquito cells each resulted in the loss of a conserved glycosylation site at Asn 153 in E. Passage in mouse brain selected for mutations at E residues 18, 54, 277, 401, and 403. Residues which altered on passaging have been localized on the three-dimensional structure of the tick-borne encephalitis virus E protein soluble fragment (F. A. Rey, et al., 1995, Nature 375, 291-298). Residues 54, 191, 202, 266, 268, and 277 map to a postulated "hinge" region between domains I and II which may be involved in fusion of flaviviruses with cell membranes. The oligosaccharide at Asn 153 also appears to be involved in flavivirus fusion. Changes in the fusion characteristics of the passaged viruses were demonstrated.

Replication of standard and defective Ross River virus in BHK cells: patterns of viral RNA and polypeptide synthesis.

Virus-specific macromolecule synthesis has been examined in BHK cells infected with Ross River virus. Unpassaged virus (R-0) and tenth-passage virus (R-10) have been compared. In infected cells R-0 generates i) 45S, 28S, 33S and 26S viral RNAs, ii) virus-specific precursor polypeptides of mol. wt. 127,000, 95,000 and 61,000 and iii) viral envelope proteins (mol. wts. 52,000 and 49,000) and nucleocapsid protein (mol. wt. 32,000). Thus in terms of virus-specific RNA and polypeptide synthesis, the replication of standard RRV is analogous to that of Semliki Forest virus and Sindbis virus. R-10 interferes with the replication of standard Ross River virus and generates large amounts of 19S and 24S defective RNA species; 45S and 26S RNA synthesis was not markedly affected. Defective RNAs are associated with RNAse-sensitive, 50S cytoplasmic particles which contain a variety of (mainly host) proteins but no nucleocapsid protein. No evidence for translation of defective RNAs was obtained. R-10 infection is also characterized by a relatively early shut down of host protein syntehsis and by a reduction in virus-specific polypeptide synthesis and nucleocapsid formation. The data suggest that defective Ross River virus interferes primarily at the translational level.

Genetic analysis of Kunjin virus isolates using HaeIII and TaqI restriction digests of single-stranded cDNA to virion RNA.

The genotypic relatedness of 15 Kunjin (KUN) virus isolates from widely separated geographic regions in Australia was examined at the molecular level. HaeIII and TaqI restriction digest profiles of cDNA transcribed from virion RNA revealed a close genetic similarity between all isolates. We estimate that the nucleotide sequence divergence between any pair of KUN isolates is probably less than 1%. We conclude that a single KUN genetic type has existed in enzootic and epizootic areas of virus activity over an extended time span. The epidemiological implications of these results are discussed.

A mouse-attenuated envelope protein variant of Murray Valley encephalitis virus with altered fusion activity.

A neutralization escape variant of Murray Valley encephalitis virus (MVE), of low neuroinvasiveness in mice and with low haemagglutination activity, had a reduced rate of replication in cultured cells during the early phase of infection compared to wild-type MVE. The variant was internalized by Vero cells at a similar rate to wild-type MVE at pH 7.4, but had reduced pH-dependent membrane fusion activity. In fusion-from-within experiments in infected mosquito (C6/36) cells, the variant had a lowered pH threshold for induction of fusion, which occurred at a reduced rate and to a lesser extent than for wild-type virus. Fusion was inhibited by monoclonal antibodies specific for envelope protein epitopes E-5 and E-8, which were implicated as determinants of fusion. These observations are discussed in relation to the regulation of MVE replication by fusion of the viral envelope with endosome membranes and, in turn, how rates of replication may affect neuroinvasion.

A comparison of the spread of Murray Valley encephalitis viruses of high or low neuroinvasiveness in the tissues of Swiss mice after peripheral inoculation.

A Murray Valley encephalitis virus (MVE) field isolate of high neuroinvasiveness (BH3479) and a neutralization escape variant of low neuroinvasiveness (BHv1) selected from BH3479 (which differ by a single amino acid at residue 277 in the envelope glycoprotein) were examined for their distribution in the tissues of weanling Swiss mice at various times after footpad inoculation. BH3479 was first detected in lymph nodes draining the inoculated limb at 24 hr postinoculation (pi) and was found in serum between 36 and 72 hr pi. BH3479 was first detected in the central nervous system (CNS) at 4 days pi and reached maximum CNS titers ( > 10(9) PFU/g) between 6 and 9 days pi. All BH3479-infected mice developed encephalitis and died before 10 days pi. In contrast, BHv1 was not detected in lymph nodes draining the footpad at any time after inoculation; BHv1 was first detected in the serum between 60 and 72 hr pi-24 hr later, and at a 20-fold lower titer than for BH3479. BHv1 was first detected in the CNS at 7 days pi 3 days later and at a 300-fold lower titer than for BH3479. After 10 days pi, BHv1 could not be isolated from the CNS or from other host tissues. Most BHv1-infected mice experienced a subclinical infection; the mortality rate from BHv1 infection was less than 1%. Both viruses appeared to enter the CNS via the olfactory lobes. BH3479 spread throughout the CNS in a rostral to caudal direction over 3-4 days. In contrast, BHv1 infection in the CNS was restricted to the olfactory lobes and adjacent structures of the forebrain.

Stimulation of dengue virus replication in cultured Aedes albopictus (C6/36) mosquito cells by the antifungal imidazoles ketoconazole and miconazole.

Replication of dengue type 3 virus in Aedes albopictus C6/36 cells was enhanced more than 50-fold by addition of the antifungal imidazole derivative ketoconazole within the first 4 h of infection. The stimulatory effect was reflected in the yield of infectious virus and in levels of viral RNA and protein synthesis. Enhanced yields were observed also for other flaviviruses, including dengue type 2 virus and Murray Valley encephalitis virus. Increased yields of dengue type 3 virus were not observed in African green monkey kidney (Vero) cells, human monocytic (U-937) cells, or cells of the mosquito Toxorhynchites amboinensis (TRA-171).

Ross River virus variants selected during passage in chick embryo fibroblasts: serological, genetic, and biological changes.

Serial passage of Ross River virus in chick embryo fibroblasts selected for virus variants altered in their reactions to neutralizing monoclonal antibodies. The E1 and E2 genes of each antigenic variant were sequenced; single nucleotide changes were found in E2 leading to amino acid substitutions at either residue 4 (Glu-->Lys) or 218 (Asn-->Lys); no changes were found in the E1 gene. Variants with the substitution at E2 residue 218 replicated less efficiently in 1-day-old mice than did the parental strain. The variant changed at E2 residue 4 showed little alteration in replication efficiency in mice. Similar genotypic or phenotypic changes were not found in virus passaged serially in human or mosquito cell lines.

Genetic differentiation of Murray Valley encephalitis virus in Australia and Papua New Guinea.

The genetic relatedness of ten Murray Valley encephalitis virus (MVE) isolates from Australia has been examined by comparing HaeIII and TaqI restriction digest profiles of cDNA to virion RNA. The isolates were from the Murray Valley region of south-eastern Australia and from the Ord River region of Western Australia and spanned a period of 23 years (1951-1974). The isolates generated closely similar restriction digest profiles. The extent of similarity suggested that the level of nucleotide sequence divergence between any pair of Australian MVE isolates is probably around 1%. The genetic homogeneity of the MVE isolates contrasts with results obtained for Ross River virus, an alphavirus, using an identical methodological approach; we propose that this difference results from the important role of birds in the life cycle of MVE. Four MVE isolates from three fatal human cases showed small genetic differences one from the other. These isolates did not have a common restriction digest profile which distinguished them from strains obtained from other sources (e.g., from mosquitoes or a heron). The data do not support the view that clinical cases of MVE infection in humans are due to a particular strain of virus although this has not been rigorously excluded. The two available MVE isolates from Papua New Guinea (PNG) were from the Sepik and Port Moresby regions. They generated HaeIII and TaqI restriction digest profiles which were different both from each other and from those of the Australian type. Genetic divergence between the two PNG isolates was estimated to be approximately 6%; divergence between either of the PNG isolates and the Australian type was greater than 6%. Our data suggest that the evolution of MVE in Australia and PNG has proceeded independently and that circulating Australian MVE strains are not systematically re-seeded from regions of endemicity in PNG. Studies on the relatedness of MVE and two close antigenic relatives, Japanese encephalitis virus (JE) and Alfuy virus (ALF), showed that the genetic relatedness between any MVE isolate and JE or ALF is less than that between the most divergent of the MVE isolates, including those from Papua New Guinea.

Ross River virus mutant with a deletion in the E2 gene: properties of the virion, virus-specific macromolecule synthesis, and attenuation of virulence for mice.

A mutant of RRV T48 the prototype strain of Ross River virus has been isolated with a 21-nucleotide deletion in the gene coding for the envelope glycoprotein E2. Direct sequencing of the 26 S subgenomic RNA, together with HaeIII and TaqI restriction digest analysis of cDNA to RNAs from cells infected with the mutant virus (RRV dE2) and with RRV T48, were consistent with the deletion being the only major alteration in the mutant genome. The E2 protein of RRV dE2 virions had a higher electrophoretic mobility than that of RRV T48 E2 protein. Neither RRV dE2 nor RRV T48 virions contained more than trace amounts of E3, the small envelope glycoprotein found in Semliki Forest virus. RRV dE2 generated small plaques on Vero cell monolayers; plaque formation was not temperature-sensitive between 32 and 41 degrees. By comparison with RRV T48 the infectivity of RRV dE2 virions was thermolabile at 50 degrees. In BHK cells RRV dE2 grew with similar kinetics to RRV T48. Rates of synthesis of 26 S RNA and 49 S RNA were higher in cells infected with RRV dE2 than in cells infected with RRV T48. Virus-specific protein synthesis and shut-down of host protein synthesis occurred 2-3 hr earlier in RRV dE2-infected cells than in cells infected with RRV T48. Minor differences between the two viruses were observed in the profiles of virus-specific proteins generated in infected cells. In day-old mice RRV dE2 induced less severe symptoms of hind leg paralysis than did RRV T48. A small increase in LD50 and average survival time was observed in RRV dE2-infected mice by comparison with RRV T48 infected mice. Peak titers reached by RRV dE2 in the hind leg muscle, brain, and blood of day-old mice were 3-4 log units less than the titers reached during infection with RRV T48. In week-old mice the differences in virulence between the two strains were magnified: RRV dE2 induced no detectable symptoms even when injected at high doses (8 X 10(6) PFU) whereas the LD50 and average survival time for RRV T48 were unchanged from those in day-old mice. Peak RRV dE2 titers in hind leg muscle, brain, and blood, respectively, were 2, 5, and 5 log units less than the corresponding titers for RRV T48. Peak muscle titers reached by RRV dE2 were similar (approximately 10(8) PFU/g tissue) in day-old mice where lethality was high and in week-old mice where the virus was avirulent.(ABSTRACT TRUNCATED AT 400 WORDS)

Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain).

The location of a major antigenic determinant involved in the neutralization of a flavivirus, yellow fever virus (YF), has been defined in terms of its position in the amino acid sequence of the E protein. Neutralization escape variants of the 17D vaccine strain of YF were selected with two neutralizing monoclonal antibodies. Nucleotide sequencing of the envelope protein genes (E and M) of the variants showed that in each variant there was a single nucleotide change in the E gene leading to a nonconservative amino acid substitution in the E protein at position 71 or 72. The changes are in a region of the E protein which is hydrophilic, rich in cysteine residues, and not conserved between flavivirus subgroups. Since the selecting monoclonal antibodies neutralize attenuated 17D and virulent Asibi strains of YF with equal efficiency (J. J. Schlesinger, M. W. Brandriss, and T. P. Monath, 1983, Virology 125, 8-17), it can be concluded that the neutralization determinant defined for 17D YF is also present in Asibi YF.

Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes.

Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.

Characterization of a major neutralization domain of Ross river virus using anti-viral and anti-peptide antibodies.

The E2 glycoprotein of the alphavirus Ross River virus (RRV) contains three defined neutralization epitopes (a, b1 and b2) with determinants located between amino acids 216 and 251 in the linear sequence (Vrati et al., 1988, Virology 162, 346-353). The antigenic structure of this region has been examined using hyperimmune mouse antiserum against RRV and antiserum against four synthetic peptides representing linear amino acid sequences in the neutralization region of E2. In plaque reduction neutralization tests using hyperimmune antiserum to RRV, an RRV mutant altered at all three neutralization epitopes was markedly more resistant than the parental virus; variants altered at single epitopes could not be distinguished in these tests. Sera from mice immunized with synthetic RRV E2 peptides conjugated to keyhole limpet haemocyanin reacted, in a direct ELISA, with the specific region of RRV represented by the peptide. The same sera did not neutralize or immunoprecipitate RRV in solution or bind to RRV in a capture ELISA. The RRV peptides did not prime mice to react to a subimmunogenic dose of RRV; they did not bind monoclonal or polyclonal antibodies to RRV. We conclude that a significant proportion of the neutralizing antibody response in mice is elicited by epitopes a, b1, and b2 of RRV E2 and that the sites to which neutralizing antibodies bind are formed by complex folding.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries.

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.