comparison of natural killer cells induced by kunjin virus and corynebacterium parvum with those occurring naturally in nude mice.natural killer (nk) cells are rapidly elicited in the spleen and peritoneal cavity of mice inoculated intravenously or intraperitoneally with live kunjin virus, and more slowly in the peritoneal cavity of mice inoculated intraperitoneally with formalin-inactivated corynebacterium parvum. nk cells induced by either agent display cytotoxicity for a similar spectrum of syngeneic, allogeneic, and xenogeneic cultured cell lines. by contrast, the cells occurring naturally in the spleen of congenitally ...1979160889
isolation of kunjin virus from a patient with a naturally acquired describe the first isolation of kunjin virus from a patient with a natural infection.19921321945
kunjin virus isolates of australia are genetically homogeneous.the genomes of 22 isolates of kunjin virus (kun) from australia were characterized and compared using rnase t1 oligonucleotide fingerprinting. the results show that all isolates belonged to one topotype, the distribution of which covered the entire australian continent. this finding is similar to that of murray valley encephalitis virus, but in contrast to the results reported for some other flaviviruses such as saint louis encephalitis virus.19892552010
nucleotide and complete amino acid sequences of kunjin virus: definitive gene order and characteristics of the virus-specified proteins.a kunjin (kun) virus cdna sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. partial n-terminal amino acid analyses of kun virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. the gene order relative to that proposed for yellow fever (yf) virus is as follows: kun 5'-c.gp20.e.gp44.p19.p10.p71.(?).p21.p98-3' yf 5'-c.prm.e.ns1.ns2a.ns2b.ns3.ns4a.ns4b. ns5-3'. the order of putative signal sequences ...19882826659
the proteins of murray valley encephalitis virus.proteins specified by murray valley encephalitis virus were labelled during virus growth in vero and in ps cells, and separated by polyacrylamide gel electrophoresis. the purified virus particle contains three proteins (v-1, v-2 and v-3) whereas the slow sedimenting haemagglutinin or virus sub-particle lacks the core protein v-2 but contains nv-2, a non-structural protein. seven non-structural proteins in addition to v-2 and v-3 were identified in infected cells. electrophoretic profiles by viru ...1975806663
the responses of the popliteal lymph node of the sheep to ross river and kunjin viruses.the responses of popliteal lymph nodes of sheep to ross river virus (rrv) or kunjin virus (kv) have been studied by monitoring the cell populations, virus titre, antibody titre and plaque-forming cell (pfc) content of the efferent lymph. in a typical primary response to subcutaneous inoculation in the lower hind limb of either rrv or kv, the flow rate of lymph increased slightly. increased cell concentrations in the lymph following inoculation caused the hourly cell outputs to rise 5.3- to 10.1- ...1976797381
possible involvement of receptors in the entry of kunjin virus into vero cells.the results obtained from electron microscopy, adsorbed and internalised virus assays and immunofluorescence studies supported that the most likely mode of entry of kunjin virus into vero cells was by receptor-mediated endocytosis. this was deduced indirectly from the time sequence of events that occurred. electron microscopy revealed that endocytosis of the virus through coated vesicles had occurred. the adsorbed and internalised virus assay and immunofluorescence studies showed that there were ...19882899998
detection of flavivirus antigens in purified infected vero cell plasma membranes.the types of kunjin virus-specified proteins present in purified vero cell plasma membrane were studied. immunofluorescence of unfixed kunjin virus-infected whole cell monolayers, indicated that two structural proteins (envelope and prm) and three non-structural proteins (ns1, 3 and 5) were found at the plasma membrane. there was no obvious progressive accumulation of the observed antigens over the time periods between 8 to 24 h p.i. thus sds-page analysis was performed using purified radiolabel ...19921331144
murray valley encephalitis in australia, 1974: antibody response in cases and community.fifty-three patients accepted on clinical grounds as cases of murray valley encephalitis (mve) in australia in 1974 were examined for antibody to mve virus. only one (who died early in the disease and whose diagnosis was confirmed by virus isolation) did not develop antibody; 13 patients showed stationary or single convalescent titres not diagnostic of recent infection, but other evidence that infection was recent was obtained in eight; 39 showed significant rise or fall in titre confirming rece ...19761071876
serological evidence of inter-epidemic infection of feral pigs in new south wales with murray valley encephalitis virus.the sera of 617 feral pigs, collected from three widely separated areas of northern and central new south wales, were examined for antibody to murray valley encephalitis (mve) virus and to ross river virus. haemagglutination-inhibition (hi) antibody was detected to mve in 58% of sera and to ross river virus in 15% of sera. neutralization tests suggested that the mve hi antibody resulted from infection with mve virus in the summers of 1971-1972 and 1972-1973 when the virus was not known to be act ...19761016127
proteins of the group b arbovirus kunjin.purified kunjin virus was disrupted with sodium dodecyl sulfate, urea, and mercaptoethanol or acetic acid. electrophoresis on 7.5% polyacrylamide gel separated four proteins of high (120,000), intermediate (65,000) and low (18,000 and 13,000) molecular weights. a "core" particle was obtained by degradation of the virion with deoxycholate at 0 c; it contained the viral ribonucleic acid and the two small structural proteins. the "envelope" material released from around the core was identified with ...19694982830
successful competition in translation by the flavivirus kunjin with poliovirus during co-infections in vero cells.infection with poliovirus effectively inhibited the translation of vero cell messengers (carrying type 1 or type 2 caps at the 5' end) and of influenza virus messengers (type 1 caps) in co-infections. in contrast, kunjin virus rna (type 1 caps) and semliki forest virus rna (a togavirus, with type 0 caps) continued to be translated in the presence of co-infecting poliovirus. translation of kunjin virus rna was also unaffected during co-infections with either influenza virus or semliki forest viru ...19902171465
positive identification of ns4a, the last of the hypothetical nonstructural proteins of flaviviruses.a radiolabeled protein migrating in sds-polyacrylamide gels near the core protein c of kunjin virus-infected cells was isolated and subjected to n-terminal amino acid sequencing. comparisons with the translation sequence deduced from the known nucleotide sequence identified a hydrophobic protein of 149 amino acids located in the polyprotein sequence between ns3 and ns4b, thus establishing its identity as ns4a with a calculated mr 16,100. the cleavage sites identified at the n- and c-termini are ...19892541547
replication strategy of kunjin virus: evidence for recycling role of replicative form rna as template in semiconservative and asymmetric replication.only three forms of kunjin virus-specified rna were isolated from cytoplasm early after the latent period (about 15 hr) viz., 44 s genomic-sized single-stranded rna, 20 s double-stranded "replicative form" (rf), and 20-28 s partially ribonuclease-resistant (about 70%) "replicative intermediate" (ri). the rf and ri were resolved by electrophoresis in aqueous-agarose gel only following licl fractionation. the ri did not enter urea-polyacrylamide gels. after denaturation of untreated or rnase-treat ...19852578239
heterogeneity among flavivirus proteins separated in slab gels.35s-methionine-labeled proteins specified in vero cells by flaviviruses were analysed by sds-phosphate electrophoresis in polyacrylamide slab gels. the clarity of the profile produced by kunjin virus permitted designation of the nonstructural proteins, and confirmed the identity of nv21/2; the profile included a protein previously designated v2 (core protein) but now named nv11/2 because it migrates perceptibly faster than v2. despite a varying background of labeled host proteins, identifiable p ...1977869717
proteins specified by togaviruses in infected aedes albopictus (singh) mosquito cells.yields of greater than 10(7) p.f.u./ml at 28 or 37 degrees c of the alphavirus sindbis and the flavivirus kunjin were obtained in the aedes albopictus (singh) cell line, the latent periods being 4 to 6 and 10 to 12 h, respectively. despite a high background of host protein synthesis, virtually all the virus-specified proteins of the flaviviruses kunjin, dengue-2 and japanese encephalitis were labelled and resolved by slab gel electrophoresis of infected and uninfected cell proteins. in contrast, ...1979479844
replication of flaviviruses: separation of membrane translation sites of kunjin virus proteins and of cell proteins. 19806251615
comparisons of the peptide maps of kunjin virus proteins smaller than the envelope protein.we analyzed the maps of [35s]methionine-labeled tryptic peptides of the kunjin virus-specified proteins nv2 1/2, nv2, v2, nv1 1/2, nv1, and v1. the peptides of nv1 1/2 are identical to those of v2, except for one peptide contained only in the latter. the maps of each of the other proteins are unique and, consequently there is no evidence of any one protein being derived from another by proteolytic cleavage. the tryptic peptide maps of the above polypeptides were also compared with those of the l ...1977916031
flavivirus infection: essential ultrastructural changes and association of kunjin virus ns3 protein with microtubules.virus-induced vesicles evolved early in the kunjin virus replication cycle around 9 to 10 h p.i. just before the end of the latent period in infected vero cells. about 2 h following the appearance of the vesicles, microtubule paracrystals were also formed. these two virus-induced structures seemed interlinked and have essential roles in kunjin virus replication. a viral protein ns3 was found to be associated with the microtubule component of the cells. when vinblastine sulphate was added to the ...19892548454
unique peptide maps of the three largest proteins specified by the flavivirus kunjin.tryptic digests of four polypeptides found in kunjin virus-infected vero cells, nv5, nv4, v3, and nv3, were compared by peptide mapping. the polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. because infection of vero cells by kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during ...1977916030
broad cross-reactivity with marked fine specificity in the cytotoxic t cell response to flaviviruses.cytotoxic t (tc) cells were generated in mice of five h-2 haplotypes against the flaviviruses kunjin and west nile (wnv). a panel of recombinant vaccinia viruses which between them expressed cdna of the entire kunjin virus genome were used to infect targets. anti-kunjin virus responses to determinants derived from non-structural proteins, especially ns3, ns4a and ns4b, were dominant in most mouse strains; usually only one class i major histocompatibility complex (mhc) restriction element was inv ...19921375278
analysis of murine major histocompatibility complex class ii-restricted t-cell responses to the flavivirus kunjin by using vaccinia virus expression.the present paper analyzes the influence of major histocompatibility complex (mhc) class ii (ir) genes on mhc class ii-restricted t-cell responses to west nile virus (wnv) and recombinant vaccinia virus-derived kunjin virus antigens and identifies the immunodominant kunjin virus antigens. generally, mice were primed by intravenous infection with wnv or kunjin virus, and their cd4+ t cells were stimulated in vitro 14 days later with wnv or kunjin virus antigens to pulse macrophage or b-cell antig ...19921349926
molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of kunjin virus subcellular extracts of kunjin virus-infected cells prepared by lysis and differential centrifugation, the viral rna polymerase, rna and proteins were associated mainly with cytoplasm. when the cytoplasmic extract (500 g supernate) of infected cells labelled for 3 h from 24 h post-infection was further fractionated by rapid centrifugation through a sucrose density gradient, all viral products were located only in dense or "heavy membrane" fractions, which contained three types of virus-induce ...19921322651
immune response in rabbits to virion and nonvirion antigens of the flavivirus kunjin.the nature of the antibodies formed in rabbits in response to the following kunjin virus antigens was examined: infectious suckling mouse brain (smb), purified virion or rapidly sedimenting hemagglutinin (rha), slowly sedimenting hemagglutinin (sha), and envelope fragments prepared from rha disrupted by 0.1 or 0.2% sodium deoxycholate (doc). the hemagglutination-inhibiting (hi) and neutralizing antibody responses to smb, rha, and large envelope fragments (0.1% doc) were remarkably uniform, antib ...1977870434
peptide mapping of envelope-related glycoproteins specified by the flaviviruses kunjin and west nile.glycoproteins detected in vero cells infected by the flaviviruses west nile and kunjin were examined by gel electrophoresis and peptide mapping. two major glycoproteins, gp66 and gp54, were observed in west nile virus-infected cells labelled for short time periods with [3h]mannose. a third glycoprotein, gp58, was present in smaller amounts. pulse-labelling experiments suggested that gp66 was a precursor of gp54. peptide mapping of [3h]leucine-labelled gp66, gp54 and the envelope glycoprotein e o ...19852983002
togavirus rna: reversible effect of urea on genomes and absence of subgenomic viral rna in kunjin virus-infected cells.electrophoretic analyses showed that no rnase-sensitive rna smaller than the genome was specified by the flavivirus kunjin in infected vero cells during the period of maximum rna and protein synthesis. in contrast, rna extracted from sindbis virus-infected cells under similar conditions included the expected 42s rna (equivalent to the genome) and the smaller 26s (interjacent) rna. treatment of the genome of both togaviruses with 12 m urea produced a reversible (possibly conformational) change; m ...1977597036
comparison of centrifugation methods for molecular and morphological analysis of membranes associated with rna replication of the flavivirus kunjin.kunjin virus-infected cells were lysed and the cytoplasmic extract was subjected to sedimentation analysis. after centrifugation at 16,000 x g for 10 min about 70% of the original rna-dependent rna polymerase (rdrp) was recovered in the pellet; most of this enzymic activity was recovered in the soluble fraction after treatment with np40 detergent. membrane fractions were prepared from cytoplasmic extracts by centrifugation in discontinuous density gradients comprising w/w or w/v sucrose solution ...19921597508
strategy of the flavivirus genome: evidence for multiple internal initiation of translation of proteins specified by kunjin virus in mammalian cells. 1977888349
t-helper cell and associated antibody response to synthetic peptides of the e glycoprotein of murray valley encephalitis virus.a battery of 16 synthetic peptides, selected primarily by computer analysis for predicted b- and t-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (e) glycoprotein of murray valley encephalitis (mve) virus. we examined all of the peptides for t-helper (th)-cell recognition and antibody induction in three strains of mice: c57bl/6, balb/c, and c3h. lymphoproliferative and interleukin-2 assays were performed on splenic t cells from mice inoculated with peptides in f ...19911832722
comparisons of togaviruses: sindbis virus (group a) and kunjin virus (group b). 19724625020
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 acti ...19863017278
experimental infection of calves with a group b arbovirus (kunjin virus). 19665939451
preliminary analysis of murine cytotoxic t cell responses to the proteins of the flavivirus kunjin using vaccinia virus expression.a series of recombinant vaccinia viruses expressing various parts of the entire kunjin virus (kun) coding region was used to analyse the cytotoxic t (tc) cell responses to kun. cba/h mice inoculated with kun or west nile virus were shown to develop responses to kun or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the tc cells in vitro with kun antigens. tc cells from cba mice showed the strongest response to target cells infect ...19911713261
involvement of microtubules in kunjin virus replication. brief report.induction of microtubulin paracrystals (pc) by kunjin (kun) virus occurred after 15 hours post-infection and were often associated with convoluted membranes (cm) and virus particles. vinblastine sulphate which disrupts microtubulin, had an inhibitory effect on the virus production when added during the viral latent period. when infected samples were extracted with triton x-100 and analysed by sds-page, four viral proteins were observed.19872825618
interaction of kunjin virus with octyl-d-glucoside extracted vero cell plasma membrane.initial experiments using whole cells have shown that there were specific and saturable interactions between kunjin (kun) virus and receptor molecules on the vero cell surfaces. solubilisation of vero cell plasma membranes with octyl-d-glucoside (og) yielded an extract which also interacted specifically with kun virus. this was proven using electron microscopy. when the virus-og-extract complex was exposed onto vero cell monolayers, no kun virus was observed to enter into the whole cells. this w ...19979015287
characterization of novel viral polyproteins detected in cells infected by the flavivirus kunjin and radiolabelled in the presence of the leucine analogue hydroxyleucine.vero cells were infected by kunjin virus and radiolabelled in the presence of the leucine analogue, threo-beta-hydroxy-dl-leucine (thl). this analogue is known to prevent preprotein processing in cell-free systems when incorporated into signal peptides. novel kunjin virus-specified proteins were detected, namely gp140, p120 and gp92; the designations for the proteins indicate their approximate mr x 10(-3) and whether they are glycosylated. the glycoproteins gp140 and gp92 were observed in cells ...19873029280
carboxy-terminal analysis of nine proteins specified by the flavivirus kunjin: evidence that only the intracellular core protein is truncated.nine proteins specified by kunjin virus were labelled with [3h]lysine and digested with carboxypeptidase b which specifically cleaves carboxy-terminal lys or arg. the theoretical amount of [3h]lysine was released from the non-structural (ns) proteins ns2a, ns2b, ns3 and ns4b, which have a common carboxy terminus lys-arg deduced from cleavage sites established in the viral polyprotein by previous n-terminal amino acid analyses. this is a flavivirus consensus site, always followed by gly, ala or s ...19892549188
ultrastructural studies of kunjin virus-infected aedes albopictus cells.ultrastructural changes in aedes albopictus cells infected with kunjin virus were characterized from 12 to 72 h post-infection. early in infection (16h), there were no prominent ultrastructural changes except for an increase in the number of vacuoles in the cytoplasm. as the infection progressed the rough endoplasmic reticulum appeared to lengthen and whorls of fibres were observed within some vacuoles. virus particles were observed in small numbers scattered in the cytoplasm between 24 to 30 h ...19873029292
arbovirus infections in sarawak: the isolation of kunjin virus from mosquitoes of the culex pseudovishnui group. 19705500097
australian x disease, murray valley encephalitis and the french connection.epidemics of a severe encephalitis occurred in eastern australia between 1917 and 1925, in which over 280 cases were reported with a fatality rate of 68%. the disease had not been described previously and was called australian x disease. the next epidemic occurred in south-east australia in the summer of 1950-51. the disease was given its name of murray valley encephalitis as this was the area from which most cases were reported. a virus was isolated by eric french in victoria, and about the sam ...19958545982
envelope protein of the flavivirus kunjin is apparently not glycosylated.the envelope protein e (formerly designated v3) of the flavivirus kunjin was not labelled with radioactive galactose, mannose or glucosamine during virus growth in vero cells. on electrophoresis through polyacrylamide gels containing sds, the envelope (e) protein migrated more rapidly than related intracellular virus-specified glycoproteins. furthermore, e had a density in cscl solution consistent with that of a protein lacking carbohydrate, and did not bind to concanavalin a-agarose. in contras ...19826279774
kunjin virus encephalomyelitis.kunjin virus encephalomyelitis is described in a 49-year-old man who presented with an episode of acute encephalitis. he developed profound bulbar, truncal and proximal muscle weakness suggestive of damage to cranial motor nuclei and anterior horn cells. this resolved very slowly. a marked rise in antibody titres to kunjin virus was shown by haemagglutination inhibition tests. specific igm antibodies were detected only against kunjin virus.19863001485
restricted translation of the genome of the flavivirus kunjin in vitro.virion rna of kunjin virus was translated in rabbit reticulocyte lysates at a rate (7000 daltons/min) approaching that observed previously in vivo. as many as 18 polypeptides were translated and shown by tryptic peptide mapping to be closely related to one another and to contain some of the elements of envelope (e) and most of the elements of core (c) proteins of kunjin virus. none of the products resolved in gels were precipitated by antiserum to purified e protein, but the larger products were ...19826294228
variation in arbovirus infection rates in species of birds sampled in a serological survey during an encephalitis epidemic in the murray valley of south-eastern australia, february 1974.there was extensive and exuberant breeding of waterbirds before and during an epidemic of arboviral encephalitis in the murray valley of south eastern australia in 1974. as estimated by haemagglutination inhibition tests on 432 bird sera collected between 4th and 13th february, 1974, infection with murray valley encephalitis virus, kunjin virus and possibly other flaviviruses was concentrated in species of the order ciconiiformes (55% positive) and pelecaniformes (41%), compared with only 5% in ...19826299259
laboratory infections with arboviruses including reports of two infections with kunjin virus. 19665928304
antiviral activity released from aedes albopictus cells persistently infected with semliki forest virus.aedes albopictus (mosquito) cells persistently infected with semliki forest virus released an agent which inhibited virus production by a. albopictus cells infected with homologous virus. inhibition of virus production was accompanied by a marked reduction in the synthesis of viral rna and viral proteins. expression of the antiviral effect was prevented by pretreatment of cells with actinomycin. no analogous antiviral activity was detected in culture fluids of a. albopictus cells persistently in ...19836312089
detection of west nile virus by the polymerase chain reaction and analysis of nucleotide sequence variation.a polymerase chain reaction (pcr) assay was developed to rapidly detect and identify west nile (wn) virus. the rna from seven isolates of wn virus from six countries and four other flaviviruses (kunjin, japanese encephalitis, st. louis encephalitis, and yellow fever viruses) was reverse-transcribed (rt) and amplified by pcr. the nucleotide sequences of the amplified products were determined by a rapid, automated dna sequencing method. the wn virus rt/pcr assay detected the target gene segment of ...19938470779
rna binding properties of core protein of the flavivirus kunjin.kunjin virus (kun) c is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. in order to study the possible association of kun c with rna in vitro, we prepared several recombinant c proteins with specific deletions, each fused at the amino-terminus to glutathione-s-transferase (gst) and expressed in e. coli. they were reacted with kun rna probes transcribed in vitro from cdna representing the 5' untranslated region (5' utr ...19968645104
phenotypic changes in the flavivirus kunjin after a single cycle of growth in an aedes albopictus cell line.the properties of kunjin virus produced during acute infections of aedes albopictus (aal) mosquito cells were compared with those of the virus progeny from the c6/36 clone of mosquito cells and from vero cells. titres of 10(8) p.f.u./ml or greater were obtained from all cells, but significant haemagglutinin activity was associated only with progeny from vero and c6/36 cells. kunjin virus from aal cells adsorbed to goose erythrocytes and blocked haemagglutination by virus from vero or c6/36 cells ...19836308133
gene order of translation of the flavivirus kunjin: further evidence of internal initiation in vivo.the rates of inactivation of synthesis of individual virus-specified proteins by ultraviolet radiation provided an estimate of the target sizes of individual viral genes. in a control experiment with semliki forest virus, the genes for the structural proteins mapped in the known sequence 5' c-pe2-e1 3', and in accordance with initiation of translation from a single site on 26-s mrna. under the same conditions, the inactivation of synthesis of seven kunjin virus-specified proteins also followed f ...19846099663
viruses recovered from mosquitoes and wildlife serum collected in the murray valley of south-eastern australia, february 1974, during an epidemic of encephalitis.pools of mosquitoes collected in the murray valley in february, 1974, during an encephalitis epidemic yielded 239 isolates of 11 distinct viruses. these included 39 isolates of mve virus, an incriminated causative agent of encephalitis in man, and 111 isolates of kunjin virus, a probable causative agent. an additional isolate of mve virus was recovered from the serum of a white-faced heron, ardea novaehollandiae. the other 9 viruses comprised the alpha-viruses ross river and sindbis, the flavivi ...19826299258
synthesis of dengue virus rna in vitro: initiation and the involvement of proteins ns3 and assay for flavivirus rna-dependent rna polymerase activity in vitro was established using extracts of vero cells infected with dengue virus type 2 (den-2) or kunjin virus (kun). rna synthesis was initiated on a template of viral replicative form (rf) and rf was converted to the replicative intermediate (ri). the rna-dependent rna polymerase complex of den-2 utilised either den-2 or kun rf as template, and similarly the kun polymerase complex utilised either den-2 or kun rf template. in additi ...19938418788
characterization of kunjin virus rna-dependent rna polymerase: reinitiation of synthesis in vitro.rna-dependent rna polymerase (rdrp) activity was characterized in a cytoplasmic extract of kunjin virus-infected vero cells at 24 hr. the activity was influenced, possibly indirectly, by the length of prior treatment of infected cells with actinomycin d; however, 6 micrograms/ml actinomycin d and 10(-5) m alpha-amanitin in the rdrp assay had no effect. the replication complex was membrane-bound and mg2+ was essential for rdrp activity. incorporation was more dependent on exogenous utp and gtp th ...19873029975
further characterization of natural killer cells induced by kunjin virus.the natural killer (nk) cell induced one to two days after kunjin virus infection of balb/c mice is cytotoxic for a wide range of syngeneic, allogeneic and xenogeneic cell lines. it is also weakly cytotoxic for some non-malignant cells including mouse fibroblasts, macrophages and thymocytes, but not lymph node cells. levels of lysis of non-tumour target cells are dependent on their genotype. furthermore, malignant cell lines may become resistant following transplantation in vivo then susceptible ...19806160839
extensive nucleotide changes and deletions within the envelope glycoprotein gene of euro-african west nile viruses.we compared the sequence of an envelope protein gene fragment from 21 temporally distinct west nile (wn) virus strains, isolated in nine african countries and in france. alignment of nucleotide sequences defined two groups of viruses which diverged by up to 29%. the first group of subtypes is composed of nine wn strains from france and africa. the austral-asian kunjin virus was classified as a wn subtype in this first group. the second group includes 12 wn strains from africa and madagascar. fou ...19979292017
phylogeny of the genus flavivirus.we undertook a comprehensive phylogenetic study to establish the genetic relationship among the viruses of the genus flavivirus and to compare the classification based on molecular phylogeny with the existing serologic method. by using a combination of quantitative definitions (bootstrap support level and the pairwise nucleotide sequence identity), the viruses could be classified into clusters, clades, and species. our phylogenetic study revealed for the first time that from the putative ancesto ...19989420202
subgenomic replicons of the flavivirus kunjin: construction and applications.several kunjin virus (kun) subgenomic replicons containing large deletions in the structural region (c-prm-e) and in the 3' untranslated region (3'utr) of the genome have been constructed. replicon rna deltame with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the c gene to nucleotide 2403 near the carboxy terminus of the e gene, inclusive) and replicon rna c20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in c to nucleotide 2403) replicated efficiently ...19978995675
australian encephalitis in western australia, review the various clinical manifestations of murray valley encephalitis (mve) or kunjin virus encephalitis in patients in western australia.19938386796
detection of flavivirus rna in infected cells using photobiotin-labelled hybridization probes.ten plasmids containing viral cdna inserts of portions of the dengue virus type 2 (den-2) or kunjin virus (kun) genomes were biotinylated using photobiotin acetate and used as probes for the detection of flavivirus rna in infected vero cells. the viral cdna inserts ranged in length from 0.19 to 2.7 kilobase pairs, and represented segments of the flavivirus genome coding for structural and nonstructural proteins. in spot hybridization assays (hybridization at 60 degrees c) with rna extracted from ...19873031110
use of a flavivirus rna-dependent rna polymerase assay to investigate the antiviral activity of selected compounds.we have developed an assay using flavivirus rna-dependent rna polymerase to test the inhibitory activity of potential antiviral agents. the effects of antiviral agents on rna synthesis were examined in this assay using extracts of vero cells infected with dengue virus type 2 or kunjin virus. several different classes of known polymerase inhibitors were tested. the synthesis of double-stranded replicative form rna was inhibited in a dose-dependent fashion in the presence of the polyoxometalate hp ...19947993077
characterization of defective viral rna produced during persistent infection of vero cells with murray valley encephalitis virus.defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal rna viruses. due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. in many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. in this pape ...19989499109
functional analysis of cell surface-expressed hepatitis c virus e2 glycoprotein.hepatitis c virus (hcv) glycoproteins e1 and e2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (er). c-terminal truncation of e2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. we demonstrate cell surface expression of a chimeric glycoprotein consisting of e2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza a virus hemaggluti ...199910400776
ultrastructure of kunjin virus-infected cells: colocalization of ns1 and ns3 with double-stranded rna, and of ns2b with ns3, in virus-induced membrane structures.the subcellular location of the nonstructural proteins ns1, ns2b, and ns3 in vero cells infected with the flavivirus kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. comparisons were also made by dual immunolabelling using antibodies to double-stranded rna (dsrna), the putative template in the flavivirus replication complex. at 8 h postinfection, the immunofluorescent patterns showed ns1, ns2b, ns3, and dsrna located in a p ...19979261387
signal peptidase cleavage at the flavivirus c-prm junction: dependence on the viral ns2b-3 protease for efficient processing requires determinants in c, the signal peptide, and prm.signal peptidase cleavage at the c-prm junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the cooh terminus of the c protein. the signal peptidase cleavage occurs efficiently in circumstances where the c protein is deleted or if the viral protease complex is present. in this study, we used cdna of murray valley encephalitis virus (mve) to examine features of the structural polyprotein which allow this reg ...19989499070
orf1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex.among the functions of the replicase of equine arteritis virus (eav; family arteriviridae, order nidovirales) are important viral enzyme activities such as proteases and the putative rna polymerase and rna helicase functions. the replicase is expressed in the form of two polyproteins (open reading frame 1a [orf1a] and orf1ab), which are processed into 12 nonstructural proteins by three viral proteases. in immunofluorescence assays, the majority of these cleavage products localized to the perinuc ...19989658116
localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication.the aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (mhv). antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3c-like protease (3clpro), the putative polymerase (pol), helicase, and a recently described protein (p22) derived from the c terminus of the open reading frame 1a protein (ct1a), were used to probe mhv-infected cells by indirect immunofluorescence (if) and electron microscopy (em). at ea ...199910438855
spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus.the flavivirus genome is a positive-strand rna molecule containing a single long open reading frame flanked by noncoding regions (ncr) that mediate crucial processes of the viral life cycle. the 3' ncr of tick-borne encephalitis (tbe) virus can be divided into a variable region that is highly heterogeneous in length among strains of tbe virus and in certain cases includes an internal poly(a) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined se ...19989499069
completion of kunjin virus rna sequence and recovery of an infectious rna transcribed from stably cloned full-length cdna.completion of the kunjin virus (kun) rna sequence showed that it is the longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. the 3' noncoding sequence of 624 nucleotides included a unique insertion sequence of 46 bases adjacent to the stop codon, but otherwise it had properties similar to those of rnas of closely related flaviviruses. a full-length kun cdna clone which could be stably propagated in escherichia coli dh5 alpha was constructed; sp6 ...19948207832
a c-terminal motif found in the beta2-adrenergic receptor, p2y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the na+/h+ exchanger regulatory factor family of pdz proteins.the na+/h+ exchanger regulatory factor (nherf) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of na+/h+ exchange. nherf contains two pdz domains, the first of which is required for its interaction with the beta2 receptor. mutagenesis studies of the beta2 receptor tail revealed that the optimal c-terminal motif for binding to the first pdz domain of nherf is d-s/t-x-l, a motif distinct from those recognized by other pdz domains. the first pdz domain o ...19989671706
mutagenesis of the ns3 protease of dengue virus type 2.the flavivirus protease is composed of two viral proteins, ns2b and ns3. the amino-terminal portion of ns3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. we have undertaken a mutational analysis of the region of ns3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. in all, 46 single-amino-acid substitution ...19989420267
subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns2a and a previous study on the replication of kunjin virus using immunoelectron microscopy (e. g. westaway, j. m. mackenzie, m. t. kenney, m. k. jones, and a. a. khromykh, 1997, j. virol. 71, 6650-6661), ns1 and ns3 were found associated with double-stranded rna (dsrna) within vesicle packets (vp) in infected vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of rna replication. ns2b and ns3 (comprising the virus-encoded protease) were colocalized within disti ...19989636360
primary viraemia responses of herons to experimental infection with murray valley encephalitis, kunjin and japanese encephalitis viruses.rufous night herons, pacific herons, little egrets and intermediate egrets were experimentally infected with murray valley encephalitis, kunjin or japanese encephalitis viruses. viraemias of at least one day's duration were detected in all birds except two intermediate egrets inoculated with a very low dose of kunjin virus and one rufous night heron inoculated with japanese encephalitis virus. there was usually a viraemia of 3 to 5 days' duration commencing on the first or second day and continu ...19836326724
primary antibody responses of herons to experimental infection with murray valley encephalitis and kunjin viruses.antibody responses of rufous night herons (nycticorax caledonicus) and little egrets (egretta garzetta) following infection with murray valley encephalitis and kunjin viruses were determined. haemagglutinin-inhibiting antibodies were first detected on day 5 or 6 after inoculation and increased rapidly, reaching maximum titres of 320 to 2560 between 10 and 20 days after inoculation. titres declined 20-320 between 60 and 120 days after inoculation, then tended to remain stationary. titres were 2- ...19836326725
transcripts from a single full-length cdna clone of hepatitis c virus are infectious when directly transfected into the liver of a chimpanzee.we have succeeded in constructing a stable full-length cdna clone of strain h77 (genotype 1a) of hepatitis c virus (hcv). we devised a cassette vector with fixed 5' and 3' termini and constructed multiple full-length cdna clones of h77 in a single step by cloning of the entire orf, which was amplified by long reverse transcriptase-pcr, directly into this vector. the infectivity of two complete full-length cdna clones was tested by the direct intrahepatic injection of a chimpanzee with rna transc ...19979238047
characterization of an autonomous subgenomic pestivirus rna an initial approach to define the requirements for the replication of bovine viral diarrhea virus (bvdv), a member of the flaviviridae family with a positive-strand rna genome, full-length genomic and subgenomic rnas were originated by in vitro transcription of diverse bvdv cdna constructs and transfected into eucaryotic host cells. rna replication was measured either directly by an rnase protection method or by monitoring the synthesis of viral protein. when full-length bvdv crna was initial ...19989499097
substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.the flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (fg loop) in the envelope (e) protein. in some flaviviruses this domain harbors the integrin-binding motif arg-gly-asp (rgd). one of us has shown earlier that host cell adaptation of murray valley encephalitis virus (mve) can result in the selection of attenuated variants altered at e protein residue asp(390), which is part of an rgd motif. here, a full-length, infectious cdna clone of mve was constructed ...200010982329
dengue virus type 1 nonstructural glycoprotein ns1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion.nonstructural glycoprotein ns1, specified by dengue virus type 1 (den-1), is secreted from infected green monkey kidney (vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species. this noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kda and a stokes radius of 64.4 a. during protein export, one of the two oligosaccharides of ns1 is processed into an endo-beta-n-acetylglucosaminidase f-resis ...199910364366
encapsidation of the flavivirus kunjin replicon rna by using a complementation system providing kunjin virus structural proteins in trans.kunjin virus (kun) replicon rna was encapsidated by a procedure involving two consecutive electroporations of bhk-21 cells, first with kun replicon rna c20dxrep (with prme and most of c deleted) and about 24 h later with a recombinant semliki forest virus (sfv) replicon rna(s) expressing kun structural proteins. the presence of kun replicon rna in encapsidated particles was demonstrated by its amplification and expression in newly infected bhk-21 cells, detected by northern blotting with a kun-s ...19989621059
glycosylation and antigenic variation among kunjin virus isolates.previous studies have found kunjin (kun) virus isolates from within australia to be genetically homogenous and that the envelope protein of the type strain (mrm61c) was unglycosylated and lacked a potential glycosylation site. we investigated the extent of antigenic variation between kun virus isolates from australia and sarawak using an immunoperoxidase assay and a panel of six monoclonal antibodies. the glycosylation status of the e protein of each virus was also determined by n glycosidase f ...19957530394
analysis of murine cd8(+) t-cell clones specific for the dengue virus ns3 protein: flavivirus cross-reactivity and influence of infecting serotype.serotype-cross-reactive dengue virus-specific cytotoxic t lymphocytes (ctl) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (dhf) during a secondary dengue virus infection. although there is no animal model of dhf, we previously reported that murine dengue virus-specific ctl responses are qualitatively similar to human dengue virus-specific ctl responses. we used balb/c mice to study the specificity of the ctl respo ...19999847344
rna virus vectors: where are we and where do we need to go? 19989788984
mutagenesis of the ns2b-ns3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing.analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded ns2b-ns3 protease at the carboxy-terminal end of the c (capsid or core) protein. cleavage at this site has been implicated in the efficient generation of the amino terminus of prm via signal peptidase cleavage. yellow fever virus has four basic residues (arg-lys-arg-arg) in the p1 through p4 positions of this cleavage site. multiple alanine substitutions were made for these residues in or ...199910482557
loss of dimerisation of the nonstructural protein ns1 of kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of ns1.the flavivirus nonstructural protein ns1 has been implicated in viral rna replication, although its precise role has not been identified. in its native state ns1 exists as a heat labile homodimer that is thought to be required for ns1 function and secretion. however, we have recently identified a cdna clone of kun virus (flsd) that replicates efficiently in cell culture but produces and secretes ns1 in monomeric form. sequence analysis of the ns1 gene in flsd revealed a single amino acid substit ...199910544130
trans-complementation analysis of the flavivirus kunjin ns5 gene reveals an essential role for translation of its n-terminal half in rna replication.recently we described rescue of defective kunjin virus (kun) rnas with small deletions in the methyltransferase and rna polymerase motifs of the ns5 gene, using bhk cells stably expressing kun replicon rna (repbhk cells) as helper (a. a. khromykh et al., j. virol. 72:7270-7279, 1998). we have now extended our previous observations and report successful trans-complementation of defective kun rnas with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. rep ...199910516033
antiapoptotic but not antiviral function of human bcl-2 assists establishment of japanese encephalitis virus persistence in cultured cells.upon infection of japanese encephalitis virus (jev), baby hamster kidney (bhk-21) and chinese hamster ovary (cho) cells were killed by a mechanism involved in apoptosis. while readily established in a variety of cell lines, jev persistence has never been successfully instituted in bhk-21 and cho cells. since stable expression of human bcl-2 in bhk-21 cells has been shown to delay jev-induced apoptosis, in this study we investigated whether jev persistence could be established in such cells. when ...19989811720
outbreak of west nile virus infection, volgograd region, russia, 1999.from july 25 to october 1, 1999, 826 patients were admitted to volgograd region, russia, hospitals with acute aseptic meningoencephalitis, meningitis, or fever consistent with arboviral infection. of 84 cases of meningoencephalitis, 40 were fatal. fourteen brain specimens were positive in reverse transcriptase-polymerase chain reaction assays, confirming the presence of west nile/kunjin virus.200111266303
markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.replication of the flavivirus kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral rna synthesis and as convoluted membranes (cm) and paracrystalline arrays (pc) containing the components of the virus-specified protease (e. g. westaway, j. m. mackenzie, m. t. kenney, m. k. jones, and a. a. khromykh, j. virol. 71:6650-6661, 1997). to determine the cellular origins ...199910516064
characterization of cell lines carrying self-replicating hepatitis c virus rnas.subgenomic selectable rnas of the hepatitis c virus (hcv) have recently been shown to self-replicate to high levels in the human hepatoma cell line huh-7 (v. lohmann, f. körner, j. o. koch, u. herian, l. theilmann, and r. bartenschlager, science 285:110-113, 1999). taking advantage of this cell culture system that allows analyses of the interplay between hcv replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 y ...200111152498
yellow fever/japanese encephalitis chimeric viruses: construction and biological properties.a system has been developed for generating chimeric yellow fever/japanese encephalitis (yf/je) viruses from cdna templates encoding the structural proteins prm and e of je virus within the backbone of a molecular clone of the yf17d strain. chimeric viruses incorporating the proteins of two je strains, sa14-14-2 (human vaccine strain) and je nakayama (je-n [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. the je envelope protein (e) retained antigenic and ...199910074160
early diagnosis of murray valley encephalitis by reverse transcriptase-polymerase chain reaction.a 4-year-old aboriginal boy developed encephalitis due to murray valley encephalitis virus (mve) following an earlier infection with kunjin virus (kun). the illness was severe, resulting in cerebral atrophy and profound physical and intellectual disability. the earlier kun infection complicated his serological profile and delayed antibody responses to mve. by contrast, the reverse transcriptase-polymerase chain reaction (rt-pcr) assay detected mve in serum 3 days after the onset of illness and 4 ...200010740807
nascent flavivirus rna colocalized in situ with double-stranded rna in stable replication complexes.incorporation of bromouridine (bru) into viral rna in kunjin virus-infected vero cells treated with actinomycin d was monitored in situ by immunofluorescence using antibodies reactive with br-rna. the results showed unequivocally that nascent viral rna was located focally in the same subcellular site as dsrna, the putative template for flavivirus rna synthesis. when cells were labeled with bru for 15 min, the estimated cycle period for rna synthesis, the nascent br-rna was not digested in permea ...199910329573
trans-complementation of flavivirus rna polymerase gene ns5 by using kunjin virus replicon-expressing bhk cells.a bhk cell line persistently expressing a kunjin (kun) virus replicon rna (repbhk, similar to our recently described me/76neo bhk cell line [a. a. khromykh and e. g. westaway, j. virol. 71:1497-1505, 1997]) was used for rescue and propagation of kun viruses defective in the rna polymerase gene (ns5). a new infectious full-length kun virus cdna clone, flsdx, prepared from our previously described cdna clone pakun (a. a. khromykh and e. g. westaway, j. virol. 68:4580-4588, 1994) and possessing app ...19989696822
recombinant yellow fever viruses are effective therapeutic vaccines for treatment of murine experimental solid tumors and pulmonary metastases.we have genetically engineered an attenuated yellow fever (yf) virus to carry and express foreign antigenic sequences and evaluated the potential of this type of recombinant virus to serve as a safe and effective tumor vaccine. live-attenuated yf vaccine is one of the most effective viral vaccines available today. important advantages include its ability to induce long-lasting immunity, its safety, its affordability, and its documented efficacy. in this study, recombinant live-attenuated (strain ...200010982366
genetic interaction of flavivirus nonstructural proteins ns1 and ns4a as a determinant of replicase function.nonstructural protein 1 (ns1) of yellow fever virus (yf) is a glycoprotein localized to extracytoplasmic compartments within infected cells. we have previously shown that ns1 can be supplied in trans and is required for viral rna replication, a process thought to occur in membrane-bound cytoplasmic complexes. here we report that the ns1 gene from a related virus, dengue virus (den), is unable to function in the process of yf rna replication. this virus-specific incompatibility leads to a lack of ...199910233920
assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively.the intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (rer). building on previous studies involving immunofluorescence (if) and cryoimmunoelectron microscopy of kunjin virus (kun)-infected cells, we sought to identify the steps involved in the assembly and maturation of kun. thus, using antibodies directed against envelope protein e in if analysis, we found the accumulation of e within regions coi ...200111602720
development of dengue virus type 2 replicons capable of prolonged expression in host part of a program to develop a dengue virus vaccine which avoids the deleterious effects of antibody dependent enhancement (ade) of infection mediated by antibodies to dengue virus structural proteins, we have begun to investigate the possibility of designing dengue vaccines based on non-structural proteins.200111580862
rubella virus replication and links to teratogenicity.rubella virus (rv) is the causative agent of the disease known more popularly as german measles. rubella is predominantly a childhood disease and is endemic throughout the world. natural infections of rubella occur only in humans and are generally mild. complications of rubella infection, most commonly polyarthralgia in adult women, do exist; occasionally more serious sequelae occur. however, the primary public health concern of rv infection is its teratogenicity. rv infection of women during th ...200011023958
nucleic acid sequence-based amplification assays for rapid detection of west nile and st. louis encephalitis viruses.the development and application of nucleic acid sequence-based amplification (nasba) assays for the detection of west nile (wn) and st. louis encephalitis (sle) viruses are reported. two unique detection formats were developed for the nasba assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (nasba-ecl assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (nasba-beacon assay). the sen ...200111724870
biological consequences of deletions within the 3'-untranslated region of flaviviruses may be due to rearrangements of rna secondary was previously reported that deletions introduced into the 3'-untranslated region (3'-utr) of dengue type 4 (den 4) virus (men, r., bray, m., clark, d., chanock, r.m., lai, c.j., 1996. den 4 virus mutants containing deletions in the 3'-noncoding region of the rna genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. j. virol. 70, 3930-3937), tick-borne encephalitis (tbe) virus (mandl, c.w., holzmann, h., meixner, t., rauscher, ...199910518708
biogenesis of the semliki forest virus rna replication complex.the nonstructural (ns) proteins nsp1 to -4, the components of semliki forest virus (sfv) rna polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. all ns proteins were associated with large cytoplasmic vacuoles (cpv), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. all ns proteins were localized by immuno-electron microscopy (em) to the li ...200111264376
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