Kevin Anderson

Evaluation in nonhuman primates of vaccines against Ebola virus.

Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.

Genetic and antigenic properties of Dobrava virus: a unique member of the Hantavirus genus, family Bunyaviridae

We examined the genetic and antigenic properties of Dobrava (DOB) virus, a hantavirus associated with severe haemorrhagic fever with renal syndrome in Europe. Cloning and sequence analyses revealed the DOB M segment to consist of 3644 nucleotides, with a coding capacity of 1134 amino acids in the virus complementary-sense RNA (cRNA). Seven potential asparagine-linked glycosylation sites were identified in the M segment gene product, one in the G2 and six in the G1 coding regions. The S segment is 1667 nucleotides long, and has a single ORF in the cRNA capable of encoding a protein of 428 amino acids. Phylogenetic comparisons of the M and S segments of DOB virus to those of other hantaviruses indicated that DOB virus is similar to, but clearly distinct from Hantaan (HTN) and Seoul (SEO) viruses. Certain G2-specific, but not G1-specific monoclonal antibodies to HTN virus reacted to the same titre with DOB and homologous viral antigen. Plaque-reduction neutralization tests indicated that, of the sera tested, only antisera to SEO virus were able to neutralize DOB virus to a titre greater than 1:10; however, this neutralization titre was eightfold lower than that observed with homologous SEO virus. The data reported here confirm that DOB virus is a unique species in the Hantavirus genus, family Bunyaviridae.

Correct sequence for the major nucleocapsid protein mRNA of respiratory syncytial virus.

A nucleotide sequence for the mRNA of the major nucleocapsid (N) protein gene of respiratory syncytial virus was reported previously (N. Elango and S. Venkatesen, 1983, Nucleic Acids Res. 11, 5941-5951). However, we have been unable to confirm part of this sequence as N mRNA-specific and suggest that the published sequence represents that of an aberrant chimeric transcript. Here we present an alternative sequence for the N mRNA and provide data supporting its authenticity. The corrected N mRNA sequence contains 1197 rather than 1427 nucleotides exclusive of poly(A), and encodes a protein of 391 rather than 467 amino acids. The calculated molecular weight for the 391-amino acid protein described by the sequence presented here is 42,600, in agreement with the molecular weight of 42,000 determined for the RS viral N protein by gel electrophoresis. In addition, we present sequence data from dicistronic RNAs that span the junction between the 1B protein and N cistrons, and the junction between the N and phosphoprotein (P) cistrons.

Endogenous origin of defective retroviruslike particles from a recombinant Chinese hamster ovary cell line

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. Continuous-flow ultracentrifugation has been used to concentrate extracellular particles from culture fluid of a recombinant CHO cell subclone for molecular characterization. Particles exhibiting reverse transcriptase activity and associated with mammalian C-type retrovirus structural proteins banded in sucrose gradients at a density characteristic of retroviruses. Examination of gradient-purified particles by electron microscopy revealed morphology and size similar to other retroviruses. Double-gradient-purified particles contained RNA which hybridized to probes for murine leukemia virus, and endogenous Chinese hamster intracisternal A-particle elements. DNA sequence analysis of a cDNA clone isolated from purified particles revealed multiple interruptions of the endonuclease reading frame, providing one possible explanation for the noninfectious nature of the observed particles. Sequences present as RNA in purified particles were also present as conserved, repetitive, provirus sequences in genomic DNA of all CHO cell lines examined and in Chinese hamster liver DNA. The observed particles are therefore likely to be the products of endogenous retroviruslike elements present in the germline of Chinese hamsters.

Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A.

An eight amino acid sequence (TELRTFSI) present in the carboxy terminal end (aa 577-584) of membrane-anchored GP, the major structural protein of Ebola virus, was identified as an H-2k-specific murine cytotoxic T cell epitope. Cytotoxic T lymphocytes (CTLs) to this epitope were induced by immunizing B10.BR mice intravenously with either irradiated Ebola virus or with irradiated Ebola virus encapsulated in liposomes containing lipid A. The CTL response induced by irradiated Ebola virus could not be sustained after the second round of in vitro stimulation of immune splenocytes with the peptide, unless the irradiated virus was encapsulated in liposomes containing lipid A. The identification of an Ebola GP-specific CTL epitope and the requirement of liposomal lipid A for CTL memory recall responses could prove to be a promising approach for developing a vaccine against Ebola virus infection.

Framework for leadership and training of Biosafety Level 4 laboratory workers

One-sentence summary for table of contents: Training should include theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experience.

Bovine respiratory syncytial virus nucleocapsid protein: mRNA sequence analysis and expression from recombinant vaccinia virus vectors

The nucleotide sequence of the mRNA encoding the nucleocapsid (N) protein of bovine respiratory syncytial (BRS) virus, strain 391-2, was determined. Recombinant vectors containing a cDNA of the complete N gene were constructed, and expression of the N protein in eukaryotic cells was demonstrated using two different vector systems. The BRS virus N mRNA was 1197 nucleotides in length, exclusive of poly(A), and had a single major open reading frame that encoded a polypeptide of 391 amino acids with a calculated M(r) of 42.6K. The nucleotide and amino acid sequences of the BRS virus N gene were compared to those of human respiratory syncytial (HRS) virus strains A2 and 18537, and to BRS virus strain A51908. The level of nucleic acid identity between the N mRNA of BRS virus 391-2 and both HRS virus subtypes was 80 to 81%, whereas the identity between the two BRS virus strains was 97%. A 93 to 94% level of identity was observed between the deduced amino acid sequences of the N protein of BRS virus 391-2 and the corresponding sequences of the two HRS virus strains. The two BRS virus N proteins differed in amino acid sequence at only three positions. Recombinant BRS virus N protein was expressed using two different vector systems: in cells from a plasmid using the vaccinia virus/T7 polymerase expression system or from a recombinant vaccinia virus. N proteins synthesized by the two vector systems migrated with an electrophoretic mobility identical to that of authentic BRS virus N protein, and were precipitated by anti-BRS virus antibodies.

BAYESIAN ESTIMATION OF THERMONUCLEAR REACTION RATES

The problem of estimating non-resonant astrophysical S-factors and thermonuclear reaction rates, based on measured nuclear cross sections, is of major interest for nuclear energy generation, neutrino physics, and element synthesis. Many different methods have been applied in the past to this problem, almost all of them based on traditional statistics. Bayesian methods, on the other hand, are now in widespread use in the physical sciences. In astronomy, for example, Bayesian statistics is applied to the observation of extra-solar planets, gravitational waves, and type Ia supernovae. However, nuclear physics, in particular, has been slow to adopt Bayesian methods. We present astrophysical S-factors and reaction rates based on Bayesian statistics. We develop a framework that incorporates robust parameter estimation, systematic effects, and non-Gaussian uncertainties in a consistent manner. The method is applied to the d(p,

Potential impact of a 2-person security rule on BioSafety Level 4 laboratory workers

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Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome.

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