Biomedical subjects

B R Miller

Publications and source records attributed to B R Miller.

At least 19 recordsLinked to original sources

PubMed2004

Deglycosylation of the NS1 protein of dengue 2 virus, strain 16681: construction and characterization of mutant viruses.

The dengue 2 virus (DENV-2) NS1 glycoprotein contains two potential sites for N-linked glycosylation at Asn-130 and Asn-207. NS1 produced in infected cells is glycosylated at both of these sites. We used site-directed mutagenesis of a DENV-2, strain 16681, full length infectious clone to create mutant viruses lacking the Asn-130, Asn-207 or both of these NS1 glycosylation sites in order to investigate the effects of deglycosylation. Ablation of both NS1 glycosylation sites resulted in unstable viruses that acquired numerous additional mutations; these viruses were not further characterized. Viruses altered at the Asn-130 site exhibited growth characteristics similar to the wild-type (WT) 16681 virus in LLC-MK(2) cells and reduced growth in C6/36 cells. Viruses mutated at the Asn-207 site achieved similar titers in LLC-MK(2) cells compared to WT, however, the appearance of cytopathic effect was delayed and growth of these viruses in C6/36 cells was also reduced compared to WT virus. The plaque size of mutant viruses altered at the Asn-130 site did not differ from that of the WT virus, while mutants altered at the Asn-207 site exhibited a reduced and mixed plaque size. Temperature sensitivity studies comparing the growth of the viruses at 37 degrees C and 39 degrees C showed no significant differences compared to the WT virus. Immunofluorescent antibody staining of infected cells showed that for WT 16681 virus or the Asn-130 site mutant viruses NS1 was located throughout the cytoplasm, however, Asn-207 site mutant virus NS1 protein appeared to be localized to the perinuclear region. Viruses deglycosylated at either site exhibited a significant reduction in mouse neurovirulence compared to the WT virus. The results of our studies indicate that glycosylation of the DENV-2 virus NS1 protein may influence NS1 protein processing/transport as well as the pathogenicity of the virus.

Aedes↗

PubMed2004

Infection patterns of o'nyong nyong virus in the malaria-transmitting mosquito, Anopheles gambiae.

Arthropod-borne alphaviruses transmitted by mosquitoes almost exclusively use culicines; however, the alphavirus o'nyong-nyong (ONNV) has the unusual characteristic of being transmitted primarily by anopheline mosquitoes. This unusual attribute makes ONNV a valuable tool in the characterization of mosquito determinants of infection as well as a useful expression system in Anopheles species. We developed a series of recombinant alphaviruses, based upon the genome of ONNV, designed for the expression of heterologous genes. The backbone genome is a full-length infectious cDNA clone of ONNV from which wild-type virus can be rescued. Additional constructs are variants of the primary clone and contain the complete genome plus a duplicated subgenomic promoter element with a multiple cloning site for insertion of heterologous genes. We inserted a green fluorescent protein (GFP) gene downstream of this promoter and used it to characterize infection and dissemination patterns of ONNV within An. gambiae mosquitoes. These experiments allowed us to identify atypical sites of initial infection and dissemination patterns in this mosquito species not frequently observed in comparable culicine infections. The utility of these ONNVs for studies in anopheline mosquitoes includes the potential for identification of vector infection determinants and to serve as tools for antimalaria studies. Viruses that can express a heterologous gene in a vector and rapidly and efficiently infect numerous tissues in An. gambiae mosquitoes will be a valuable asset in parasite-mosquito interaction and interference research.

Alphavirus↗

PubMed2003

Combination of transfection agents and magnetic resonance contrast agents for cellular imaging: relationship between relaxivities, electrostatic forces, and chemical composition.

The purpose of this study was to investigate the changes in electrostatic and magnetic resonance (MR) properties observed when MR contrast agents (CAs) (Feridex, MION-46L, or G5-dendrimer-DOTA-Gd) are combined with transfection agents (TAs) under various conditions for use as a CA-TA complex basis for cellular labeling and MRI. CAs were incubated with various classes of TAs for 0-48 hr in solutions of varying concentrations and pH values. NMR relaxation rates (1/T(1), 1/T(2)), MRI and zeta potential (ZP) of CA-TA solutions were measured. TAs decreased the 1/T(1) and 1/T(2) of G5-DOTA-Gd, Feridex, and MION-46L by 0-95%. Altering the pH of G5-DOTA-Gd-TA decreased the T(1)-weighted signal intensity (SI) on MRI from 0 to 78%. Measured ZP values for G5-DOTA-Gd, Feridex, and MION-46L were -51, -41, and -2.0 mV, respectively. The TA LV had a negative ZP, while the other TAs had ZPs ranging from +20 to +65 mV. The alteration of the ZP and NMR relaxivities of the MR CAs, Feridex, MION-46L, and G5-DOTA-Gd by TAs has been demonstrated. These results enhance our understanding of the relationship between electrostatic and MR properties.

Cells, Cultured↗

PubMed2003

Isolation of a new flavivirus related to cell fusing agent virus (CFAV) from field-collected flood-water Aedes mosquitoes sampled from a dambo in central Kenya.

Cell fusing agent virus (CFAV) is an RNA insect virus that was isolated from a line of Aedes aegypti mosquito cells and has been assigned to the family Flaviviridae, genus Flavivirus. We report here the first isolation of a CFA-like virus from field-collected mosquitoes. Mosquito larvae and pupae were sampled from flooded dambos in Central Province, Kenya during the short rain season of 1999. Specimens were reared to adults, identified and pooled by species and were tested for the presence of virus. Two virus isolates were obtained from two pools of Aedes macintoshi mosquitoes. The virus isolates replicated only in invertebrate cells in culture and not in vertebrate cells or in mice. The virus isolates did not antigenically cross-react with known arboviruses but were identified to family by reverse-transcriptase polymerase chain reaction (RT-PCR) performed using primers specific to alphaviruses, bunyaviruses and flaviviruses; only the flavivirus-specific primers produced a DNA fragment of the expected size. Nucleic acid sequencing of this fragment showed the two isolates to be nearly identical. Comparison of sequences to the GenBank database using BLAST identified the virus as most closely related to CFAV. Results from cross-neutralization tests suggested that, although the BLAST search indicated homology to CFAV, the virus isolated represented a new insect flavivirus. Detailed characterization of this new virus, described in Crabtree et al. [7], further supports this finding. We propose this new flavivirus be designated Kamiti River virus (KRV). This is the first isolation of a CFA-like virus from field-collected mosquitoes and indicates the presence of this group of viruses in nature.

Aedes↗

PubMed2003

Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus.

We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5' and 3' non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.

Aedes↗

PubMed2003

Growth characteristics of the veterinary vaccine candidate ChimeriVax-West Nile (WN) virus in Aedes and Culex mosquitoes.

In 1999 West Nile (WN) virus was introduced to North America where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (Diptera: Culicidae). Increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. 'ChimeriVax-West Nile(vet)' is being developed for use as a veterinary vaccine to protect against WN infection. This chimeric virus contains the pre-membrane (prM) and envelope (E) genes from the wild-type WN NY99 virus (isolated from a flamingo in New York zoo during the 1999 WN epidemic) in the backbone of yellow fever (YF) 17D vaccine virus. Replication kinetics of ChimeriVax-WN(vet) virus were evaluated in mosquito cell culture (Aedes albopictus C6/36), in WN vector mosquitoes [Culex tritaeniorhynchus Giles, Cx. nigripalpus Theobald and Cx. quinquefasciatus Say (Diptera: Culicidae)] and in YF vectors [Aedes aegypti (L) and Ae. albopictus (Skuse)], to determine whether these mosquitoes become infected through feeding on a viraemic vaccine, and their potential infectivity to transmit the virus. Growth of ChimeriVax-WN(vet) virus was found to be restricted in mosquitoes, compared to WN virus in Ae. albopictus C6/36 cells. When inoculated intrathoracically, ChimeriVax-WN(vet) and YF 17D viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus; replication was very restricted compared to the wild-type WN virus in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. When fed on hanging drops with ChimeriVax-WN(vet) virus (7.7 log10 PFU/mL), none of the Culex mosquitoes became infected; one Ae. albopictus and 10% of the Ae. aegypti became infected, but the titre was very low and virus did not disseminate to head tissue. ChimeriVax-WN(vet) virus had a replication profile similar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These results suggest that the natural mosquito vectors of WN and YF viruses, which may incidentally take a bloodmeal from a vaccinated host, will not become infected with ChimeriVax-WN(vet) virus.

Aedes↗

PubMed2001

Evidence for substantial variations of atmospheric hydroxyl radicals in the past two decades.

The hydroxyl radical (OH) is the dominant oxidizing chemical in the atmosphere. It destroys most air pollutants and many gases involved in ozone depletion and the greenhouse effect. Global measurements of 1,1,1-trichloroethane (CH3CCl3, methyl chloroform) provide an accurate method for determining the global and hemispheric behavior of OH. Measurements show that CH3CCl3 levels rose steadily from 1978 to reach a maximum in 1992 and then decreased rapidly to levels in 2000 that were lower than the levels when measurements began in 1978. Analysis of these observations shows that global OH levels were growing between 1978 and 1988, but the growth rate was decreasing at a rate of 0.23 +/- 0.18% year(-2), so that OH levels began declining after 1988. Overall, the global average OH trend between 1978 and 2000 was -0.64 +/- 0.60% year(-1). These variations imply important and unexpected gaps in current understanding of the capability of the atmosphere to cleanse itself.

Journal Article↗

PubMed2001

Phylogeny of Thogoto virus.

Thogoto virus is a tick-borne member of the family Orthomyxoviridae. Previously, based on the similarity in antigenic relationship by cross-neutralization test, all virus strains were concluded to have derived from the same origin. In this study, we obtained partial gene sequences of 4 genes (PB1-like protein, PA-like protein, glycoprotein, and nucleoprotein) of 8 Thogoto virus strains isolated in Africa, Asia, and Europe and studied the genetic variation and phylogeny. Unrooted phylogenetic trees created by both neighbor-joining and maximum likelihood methods based on nucleotide and amino acid sequences for 4 genes were mostly similar and revealed two lineages, Euro-Asian and African. Intra-lineage nucleotide sequence variation was greater in the Euro-Asian lineage than in the African lineage for all 4 genes. Furthermore, for the strains of Euro-Asian lineage, variations for two genes associated with RNA-dependent RNA polymerase activities were greater than those for glycoprotein or nucleoprotein gene, based on both nucleotide and amino acid sequence differences as well as on synonymous and nonsynonymous differences, indicating greater mutation rates for the polymerase activity genes in these strains.

Base Sequence↗

PubMed2001

Studies on feeding peanut meal as a protein source for broiler chickens.

Four experiments were conducted to compare the performance of broilers fed soybean meal (SBM) versus peanut meal (PNM) as protein sources. Ross x Ross 208 broiler chickens were placed in battery brooders (Experiments 1 to 3, four replicates of 8 chicks per treatment) and floor pens (Experiment 4, four replicates of 34 chicks per treatment). In Experiment 1, addition of 0, 0.1, 0.2, and 0.3% Thr to a corn-PNM-based diet increased 0 to 18 d BW gain (BWG; 0.374c vs. 0.495b vs. 0.508b vs. 0.508b kg, respectively) and decreased feed conversion ratio (FCR; 2.09c vs. 1.63b vs. vs. 1.54b vs. 1.54b g/g, respectively) compared to the corn-SBM-based control diet (BWG = 0.593a and FCR = 1.36a). In Experiment 2, diets were formulated with the same amino acid minimums, and as the percentage of PNM increased in the diets (0, 10, 20, and 32%), BWG decreased (0.560a vs. 0.532a vs. 0.521a vs. 0.458b kg, respectively) and FCR increased (1.72b vs. 1.71b vs. 1.79bc vs. 1.86c g/g, respectively). In Experiment 3, addition of Thr to a corn-PNM-based diet increased BWG (-Thr = 0.284c vs. +Thr = 0.397b kg) and decreased FCR (-Thr = 1.60b vs. +Thr = 1.54b g/g). The BWG and FCR were best for the corn-SBM-based control diet (0.499a kg and 1.38a g/g, respectively). In Experiment 4, during the growing period (18 to 42 d), significant interactions occurred between protein source (PNM vs. SBM) and protein level (16 and 20% vs. 24%) for BW and FCR but not for carcass, breast, or leg quarter yield or fat pad weights (P < 0.05) at 42 d of age. Technical (not economic) performance of birds fed PNM was similar to SBM at the highest protein levels fed. PNM could be used as a protein source for broilers under appropriate economic conditions.

Amino Acids↗

PubMed2001

Vagal nerve stimulation for status epilepticus.

Refractory generalized convulsive status epilepticus in a 13-year-old boy was halted by left vagal nerve stimulation. Over the next 1.5 years, seizures have continued at a rate and severity which is significantly better than it had been in the year before insertion of the stimulator.

Adolescent↗

PubMed2000

Natural methyl bromide and methyl chloride emissions from coastal salt marshes.

Atmospheric methyl bromide (CH3Br) and methyl chloride (CH3Cl), compounds that are involved in stratospheric ozone depletion, originate from both natural and anthropogenic sources. Current estimates of CH3Br and CH3Cl emissions from oceanic sources, terrestrial plants and fungi, biomass burning and anthropogenic inputs do not balance their losses owing to oxidation by hydroxyl radicals, oceanic degradation, and consumption in soils, suggesting that additional natural terrestrial sources may be important. Here we show that CH3Br and CH3Cl are released to the atmosphere from all vegetation zones of two coastal salt marshes. We see very large fluxes of CH3Br and CH3Cl per unit area: up to 42 and 570 micromol m(-2) d(-1), respectively. The fluxes show large diurnal, seasonal and spatial variabilities, but there is a strong correlation between the fluxes of CH3Br and those of CH3Cl, with an average molar flux ratio of roughly 1:20. If our measurements are typical of salt marshes globally, they suggest that such ecosystems, even though they constitute less than 0.1% of the global surface area, may produce roughly 10% of the total fluxes of atmospheric CH3Br and CH3Cl.

Atmosphere↗

PubMed2000

Genetic differentiation in the yellow fever virus vector, Aedes simpsoni complex, in Africa: sequence variation in the ribosomal DNA internal transcribed spacers of anthropophilic and non-anthropophilic populations.

Mosquitoes of the Aedes simpsoni complex are important vectors of yellow fever virus in Africa. We examined the ribosomal DNA sequence divergence in the internal transcribed spacer regions (ITS-1 and ITS-2) for populations of mosquitoes that were determined to be anthropophilic or non-anthropophilic in their bloodmeal host preference. A neighbour-joining tree produced two clades: one contained all of the individual mosquitoes from anthropophilic populations and the other contained all of the individual mosquitoes from non-anthropophilic populations. There was no segregation of the taxa within each of the two clades based on geographical origin. The data suggest the exisf'tence of two distinct species of Ae. simpsoni s.l. in Uganda that correlates with their host blood-feeding preference. The current taxonomic status of the complex is discussed in relation to these findings.

Aedes↗

PubMed2000

Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay.

The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins. We have developed a novel two-step "organelle trap" assay involving affinity selection and in vitro pore assembly. In the first step, soluble proteins derived from Xenopus egg extracts are applied to a column containing a ligand of interest. The bound proteins are then tagged by biotinylation and eluted. In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by their biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency conditions, three additional proteins bind to WGA-Sepharose and are revealed by the organelle trap assay. We identified all three as partner nucleoporins. Two were discovered to be Xenopus Nup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188. This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via the N-acetylglucosamine-modified nucleoporins. A gene encoding human Nup188 was also identified. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein-protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore.

Acetylglucosamine↗

PubMed2000

Identification of Culex vishnui subgroup (Diptera: Culicidae) mosquitoes from the Ryukyu Archipelago, Japan: development of a species-diagnostic polymerase chain reaction assay based on sequence variation in ribosomal DNA spacers.

The Culex vishnui subgroup includes three important vectors of Japanese encephalitis virus, Culex tritaeniorhynchus Giles, Cx. pseudovishnui Colless, and Cx. vishnui Theobald, all of which occur in the Ryukyu Archipelago, Japan. Although these three species have been shown to be vectors of JE virus in many areas of Southeast Asia, it is not yet known what role each plays in the transmission of the virus in this region. Reliable identification of adult, field-collected specimens is a critical component in epidemiological studies of virus transmission. Mosquitoes in the Cx. vishnui subgroup can be reliably identified in the larval stage. However, because females of these species are very similar, it is difficult to distinguish among them using morphology. We developed a polymerase chain reaction (PCR) assay for the identification of these species. Three species-specific primers were developed for the PCR assay based on a comparative analysis of the nucleotide sequence of the first internal transcribed spacer (ITS1) in the ribosomal DNA gene array. The primers, CT2REV, CP1REV, and CV1REV were designed to amplify a single DNA fragment each from Cx. tritaeniorhynchus, Cx. pseudovishnui, and Cx. vishnui, respectively, when paired with a single forward primer that is complementary to the highly conserved 18S rDNA gene. The amplified fragments were separated easily and identified on an agarose gel to facilitate species identification.

Animals↗

PubMed2000

First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley province, Kenya.

West Nile virus is a mosquito borne flavivirus endemic over a large geographic area including Africa, Asia, and the Middle East. Although the virus generally causes a mild, self-limiting febrile illness in humans, it has sporadically caused central nervous system infections during epidemics. An isolate of West Nile virus was obtained from a pool of four male Culex univittatus complex mosquitoes while we were conducting an investigation of Rift Valley fever along the Kenya-Uganda border in February-March 1998. This represents the first field isolation of West Nile virus from male mosquitoes and strongly suggests that vertical transmission of the virus occurs in the primary maintenance mosquito vector in Kenya. A phylogenetic analysis of the complete amino acid sequence of the viral envelope glycoprotein demonstrated a sister relationship with a Culex pipiens mosquito isolate from Romania made in 1996. This unexpected finding probably reflects the role of migratory birds in disseminating West Nile virus between Africa and Europe.

Amino Acid Sequence↗

PubMed2000

Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax-JE (YF/JE SA14--14--2), in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes.

The Japanese encephalitis (JE) virus vaccine candidate, ChimeriVax-JE, which consists of a yellow fever (YF) 17D virus backbone containing the prM and E genes from the JE vaccine strain JE SA14--14--2, exhibits restricted replication in non-human primates, producing only a low-level viremia following peripheral inoculation. Although this reduces the likelihood that hematophagous insects could become infected by feeding on a vaccinated host, it is prudent to investigate the replication kinetics of the vaccine virus in mosquito species that are known to vector the viruses from which the chimera is derived. In this study ChimeriVax-JE virus was compared to its parent viruses, as well as to wild-type JE virus, for its ability to replicate in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes. Individual mosquitoes were exposed to the viruses by oral ingestion of a virus-laden blood meal or by intrathoracic (IT) virus inoculation. ChimeriVax-JE virus did not replicate following ingestion by any of the three mosquito species. Additionally, replication was not detected after IT inoculation of ChimeriVax-JE in the primary JE virus vector, Cx. tritaeniorhynchus. ChimeriVax-JE exhibited moderate growth following IT inoculation into Ae. aegypti and Ae. albopictus, reaching titers of 3.6-5.0 log(10) PFU/mosquito. There was no change in the virus genotype associated with replication in mosquitoes. Similar results were observed in mosquitoes of all three species that were IT inoculated or had orally ingested the YF 17D vaccine virus. In contrast, all mosquitoes either IT inoculated with or orally fed wild-type and vaccine JE viruses became infected, reaching maximum titers of 5.4-7.3 log(10) PFU/mosquito. These results indicate that ChimeriVax-JE virus is restricted in its ability to infect and replicate in these mosquito vectors. The low viremia caused by ChimeriVax-JE in primates and poor infectivity for mosquitoes are safeguards against secondary spread of the vaccine virus.

Aedes↗

PubMed2000

Purification of the vertebrate nuclear pore complex by biochemical criteria.

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of approximately 120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin beta, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals approximately 40-50 distinct protein bands.

Animals↗

PubMed1999

Molecular and biological changes associated with HeLa cell attenuation of wild-type yellow fever virus.

Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.

Animals↗