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.

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).

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.

Yellow fever virus envelope protein has two discrete type-specific neutralizing epitopes.

Two monoclonal antibody neutralization resistant (MAbR) variants of the yellow fever (YF) 17D-204 vaccine virus strain were selected using YF type-specific MAb B39. These B39R variants were compared with the variant virus selected by Lobigs et al. (Virology 161, 474-478, 1987) using a second YF-type specific MAb (2E10) which mapped to amino acid position 71/72 in the envelope (E) protein. Neutralization assays with a panel of MAbs suggested that these two YF-type-specific epitopes are located in two discrete regions of the folded E protein. Each of the B39R variants had a single nucleotide mutation which encoded an amino acid substitution at either position E-155 or E-158. Thus, YF type-specific epitopes map to both domain I (B39) and II (2E10) of the YF virus E protein. The B39 defined epitope represents the first flavivirus neutralizing epitope localized to this region of domain I of the E protein.

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.

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.

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.

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.

Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice.

Neutralization escape variants of Murray Valley encephalitis virus were selected using a type-specific, neutralizing, and passively protective anti-envelope protein (E) monoclonal antibody (4B6C-2) which defines epitope E-1c. Nucleotide sequence analysis revealed single nucleotide changes in the E genes of 15 variants resulting in nonconservative amino acid substitutions in all cases. One variant had a three-nucleotide deletion in the E gene which resulted in loss of serine at residue 277. Changes were clustered into two separate regions of the E polypeptide (residues 126-128 and 274-277), indicating that E-1c is a discontinuous epitope. One variant (BHv1), altered at residue 277 (Ser-->Ile), failed to hemagglutinate across the pH range 5.5-7.5, in contrast to parental virus and the other escape variants which hemagglutinated at an optimal pH of 6.6. BHv1 was also of reduced neuroinvasiveness in 21-day-old mice following intraperitoneal inoculation compared to the other viruses. Parental virus and the neutralization escape variants grew equally well in both vertebrate and invertebrate cell cultures, indicating that the reduced neuroinvasiveness of BHv1 was not due to a major abnormality of replication.

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 and biological differentiation of dengue 3 isolates obtained from clinical cases in Java, Indonesia, 1976-1978.

Previous epidemiological, virological and clinical studies have documented a series of outbreaks of dengue fever and dengue haemorrhagic fever/dengue shock syndrome which occurred in Java, Indonesia in 1976-1978. In the current study we compare growth characteristics in cell culture, and nucleotide sequence data for the viral prM and E genes, of five low passage DEN-3 isolates obtained during these epidemics from clinically defined cases. All isolates had the same passage history: human sera were passed twice in mosquitoes and three times in a mosquito cell line (Aedes albopictus, C 6/36 cells). Growth differences were observed between individual isolates in Vero cells; growth differences were not observed in C 6/36 cells. Nucleotide sequencing of the prM and E gene region indicated that no two isolates were identical (sequence divergence ranged from 0.4 to 1.6% in pairwise comparisons) but that they were closely enough related to present a single genetic type. There were one or two differences in deduced amino acid sequence in E between isolates. Differences were at residues 65, 187, 298 or 443. One isolate differed from all others at residue 16 in the M protein. No relationship was apparent between the amino acid sequence of M or E and the nature of the disease profile, the year of isolation or the geographic region of isolation. The isolates showed 3.5 to 4.4% nucleotide sequence divergence from the highly-adapted H 87 prototype, isolated in the Philippines in 1956. The isolates showed a total of twelve common amino acid differences in prM and E proteins from H 87. Ten of these twelve residues were at positions which differed between the four dengue serotypes. Two differences (at residues 37 in M and 293 in E) were at positions which are conserved in sequence between the four dengue serotypes. The data are discussed in relation to the dengue outbreaks in Java in the period 1976-1978.

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.

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.

Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein.

Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavirvirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the putative proteolytic activity of NS3, segments of the yellow fever virus genome were subcloned into plasmid transcription/translation vectors and cell-free translation products were characterized. The results suggest that a protease activity encoded within NS2B and the N-terminal one-third of yellow fever virus NS3 is capable of cis-acting site-specific proteolysis at the NS2B-NS3 cleavage site and dilution-insensitive cleavage of the NS2A-NS2B site. Site-directed mutagenesis of the His-53, Asp-77, and Ser-138 residues of NS3 that compose the proposed catalytic triad implicates this domain as a serine protease. Infectious virus was not recovered from mammalian cells transfected with RNAs transcribed from full-length yellow fever virus cDNA templates containing mutations at Ser-138 (which abolish or dramatically reduce protease activity in vitro), suggesting that the protease is required for viral replication.

Sequence of the 3' half of the Murray Valley encephalitis virus genome and mapping of the nonstructural proteins NS1, NS3, and NS5.

We have determined the nucleotide sequence of the 3'-terminal half of the RNA genome of Murray Valley encephalitis virus (MVE) using seven overlapping cDNA clones; an estimated 80-90 nucleotides at the extreme 3'-end remain to be sequenced. In conjunction with previous sequence data for the 5' half (16), we can conclude that the MVE genome contains a long open reading frame of 10,302 nucleotides that encodes a polyprotein of 3434 residues. Comparison of the MVE deduced amino acid sequence with that of other flaviviruses shows that MVE is most closely related to Japanese encephalitis virus, consistent with serological studies. Using N-terminal amino acid sequence analysis, three nonstructural proteins (NS1, NS3, and NS5) have been identified and mapped on the MVE genome. MVE NS3 contains sequence motifs suggesting that its amino terminus may function as a serine protease. The central region of NS3 (in the linear amino acid sequence) has motifs that are found in NTP-binding proteins and helicases. MVE NS5 contains a conserved Gly-Asp-Asp sequence that is thought to be essential for RNA-dependent RNA polymerases.

Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence.

We have passaged the prototype strain of Murray Valley encephalitis virus in SW13 (human) cells, sequenced the E and M genes, and examined the virulence of the passaged virus for 21-day-old mice following intracranial and intraperitoneal inoculation. Six independent passage series were carried out: four in the presence of mouse hyperimmune ascitic fluid and two without antibody. Changes were observed in the E protein deduced amino acid sequence for each of the six 10th passage stocks sequenced. Eleven changes were observed in total for the six stocks sequenced; these were at residues 117, 118, 390, 423, and 460. Nine of the changes were nonconservative. Five of the six passaged variants were altered at Asp 390 which is part of an Arg-Gly-Asp (RGD) sequence. This change resulted from adaptation to SW13 cells rather than from antibody selection. The RGD sequence (and residue 423) falls within a region which is highly conserved between flaviviruses and is strongly hydrophilic. All five variants which were altered at Asp 390 were attenuated in 21-day-old mice following i.p. inoculation. We propose that the domain of E encompassing the RGD sequence is an important determinant of flavivirus pathogenicity.

Genetic and phenotypic studies on Ross River virus variants of enhanced virulence selected during mouse passage.

We have passaged Ross River virus (RRV) in mice to generate variants with increased mouse virulence and attempted to relate changes in virulence to genome sequence changes. RRV NBO (zero passage in mice) is a plaque-purified clone of the mouse-avirulent strain RRV NB5092, and is of low virulence for day-old mice. During RRV NBO replication in infant mice, its virulence for day-old mice increased markedly with time. By 7 days postinfection the LD50 value of harvested virus (passage level one) was congruent to 10(4)-fold less than that of the parental virus. No further decrease in LD50 followed 10 serial passages in infant mice. However, 10th passage level virus showed increased clinical effects in week-old mice by comparison with virus from passage levels one and two. The growth kinetics of RRV variants in mice suggested that the rate and extent of RRV replication in the brain tissue determined the enhanced mouse virulence of serially passaged virus. Seven out of eight independently passaged, 10th passage level variants had changes in the E2 gene leading to one or two amino acid substitutions. The changes were at residues 212, 232, 234, 251, 341, 27 and 172, and 72 and 134 in these variants; all changes except two were nonconservative. Residues 212, 234, and 251 form part of a neutralization determinant in RRV. Changes in epitope b2 (which includes amino acids 246, 248, and 251) alter the kinetics of RRV entry into cells (P. Kerr, R. C. Weir, and L. Dalgarno, unpublished data). First and second passage level virus of enhanced virulence was unchanged in E2 or E1 gene sequences from RRV NBO. However, 1st, 2nd, and 10th passage level virus induced higher levels of virus-specific RNA synthesis than did RRV NBO in cultured BHK cells. We propose a model for the mechanism of virulence enhancement on passaging RRV NBO in mice.

Murray Valley encephalitis virus field strains from Australia and Papua New Guinea: studies on the sequence of the major envelope protein gene and virulence for mice.

We have compared the nucleotide sequence of the gene encoding the major envelope (E) protein of a number of Murray Valley encephalitis virus (MVE) isolates from Australia and Papua New Guinea (PNG). The isolates, from widely separated geographic regions, were from four fatal human cases, a heron, and six mosquito pools and covered a period of 25 years. The sequences of the Australian strains were notable for their similarity, showing not more than 1.7% nucleotide sequence divergence in pairwise comparisons. There was 6.8% divergence in the E gene between the two available strains from PNG, and 9-10% divergence between each of the PNG strains and the Australian prototype. These data are consistent with previous conclusions based on HaeIII restriction digest analysis of cDNA to virion RNA (M. Lobigs, I. D. Marshall, R. C. Weir, and L. Dalgarno, 1986, Aust. J. Exp. Biol. Med. Sci. 64, 571-585). We conclude that a single MVE genetic type exists in Australia. Separate foci of MVE evolution appear to exist in PNG, generating greater strain variation. For all MVE isolates the deduced length of the E protein was 501 amino acids. The E protein differed at no more than three positions between any two Australian strains. The PNG strains differed from the Australian strains at 6-11 residues depending on the virus pair. Differences in amino acid sequence did not occur at a position corresponding to a previously demonstrated neutralization determinant in yellow fever virus (M. Lobigs, L. Dalgarno, J. J. Schlesinger, and R. C. Weir, 1987, Virology 161, 474-478). Thus selection for neutralization resistance may not be a major evolutionary pressure in the field situation. In comparisons between the E protein amino acid sequence of the prototype strain and those of a number of other MVE strains, 7 out of 14 differences were at residues seen at the corresponding position for Japanese encephalitis virus (JE), consistent with the close serological relationship of MVE and JE. Five Australian MVE strains and two from PNG were tested for virulence by comparing LD50 values after intraperitoneal and intracranial inoculation of 21-day-old mice; all strains were virulent by this test.