Production and characterization of guinea pig IL-5 in baculovirus-infected insect cells.

To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.

Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines.

We have analyzed the biophysical and pharmacological properties of five cloned K+ (Kv) channels (Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv3.1) stably expressed in mammalian cell lines. Kv1.1 is biophysically similar to a K+ channel in C6 glioma cells and astrocytes, Kv1.3 and Kv3.1 have electrophysiological properties identical to those of the types n and l K+ channels in T cells, respectively, and Kv1.5 closely resembles a rapidly activating delayed rectifier in the heart. Each of these native channels may be formed from the homomultimeric association of the corresponding Kv subunits, and pharmacological compounds that selectively modulate them may be useful for the treatment of neurological, immune, and cardiac disorders. The cell lines described in this report could be used to identify such drugs and we have therefore embarked on a pharmacological characterization of the five cloned channels. The compounds tested in this study include 4-aminopyridine, capsaicin, charybdotoxin, cromakalim, dendrotoxin, diltiazem, D-sotalol, flecainide, kaliotoxin, mast cell degranulating peptide, nifedipine, noxiustoxin, resiniferatoxin, and tetraethylammonium.

Sequence analysis of the Ebola virus genome: organization, genetic elements, and comparison with the genome of Marburg virus.

Sequence analysis of the second through the sixth genes of the Ebola virus (EBO) genome indicates that it is organized similarly to rhabdoviruses and paramyxoviruses and is virtually the same as Marburg virus (MBG). In vitro translation experiments and predicted amino acid sequence comparisons showed that the order of the EBO genes is: 3'-NP-VP35-VP40-GP-VP30-VP24-L. The transcriptional start and stop (polyadenylation) signals are conserved and all contain the sequence 3'-UAAUU. Three base intergenic sequences are present between the NP and VP35 genes (3'-GAU) and VP40 and GP genes (3'-AGC), and a large intergenic sequence of 142 bases separates the VP30 and VP24 genes. Novel gene overlaps were found between the VP35 and VP40, the GP and VP30, and the VP24 and L genes. Overlaps are 20 or 18 bases in length and are limited to the conserved sequences determined for the transcriptional signals. Stem-and-loop structures were identified in the putative (+) leader RNA and at the 5' end of each mRNA. Hybridization studies showed that a small second mRNA is transcribed from the glycoprotein gene, and is produced by termination of transcription at an atypical polyadenylation signal located in the middle of the coding region. The predicted amino acid sequence of the glycoprotein contains an N-terminal signal peptide sequence, a hydrophobic anchor sequence, and 17 potential N-linked glycosylation sites. Alignment of predicted amino acid sequences showed that the structural proteins of EBO and MBG contain large regions of homology despite the absence of serologic cross-reactivity.

Evaluation of the polymerase chain reaction for diagnosis of Lassa virus infection.

We evaluated the polymerase chain reaction (PCR) and hybridization procedures for diagnosis of Lassa fever. Primers were derived from a region of the small RNA segment of Lassa virus coding for the glycoprotein. Serum samples stored for a 14-year period from patients in Sierra Leone, West Africa were examined retrospectively. Blinded samples were then tested prospectively. Eighty-eight virus isolation-negative control sera were negative by PCR and hybridization. In the retrospective study, virus was isolated from 51 of 98 specimens from patients with Lassa fever, and 33 of these were positive for Lassa virus RNA by PCR, and 42 by PCR and hybridization. Fifteen were positive by PCR and hybridization but isolation-negative, and nine were positive by isolation but PCR/hybridization-negative. Thirty-two were negative by all methods (sensitivity by PCR/hybridization compared with virus isolation 0.82, specificity 0.68). In a prospective blinded study of 195 patient sera, 51 were positive by PCR and virus isolation, and 24 were PCR positive but virus isolation-negative (sensitivity 0.66, specificity 0.71). After hybridization, 66 virus isolation-positive sera were positive. The sensitivity was 0.86 and the specificity was 0.59, and the probability of false-positive results compared with virus isolation was 32%, (chi 2 = 21.9, by McNemar's test). Since some specimens may not have contained viable virus, we re-analyzed the data of individual patients using laboratory-confirmed case definitions for Lassa fever. All specimens from patients in whom Lassa fever was excluded by serologic tests were negative by PCR/hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.

Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein.

A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbs) in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, G1 and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA).

Simultaneous expression of the Lassa virus N and GPC genes from a single recombinant vaccinia virus.

A new transfer vector was constructed that directs the insertion of two heterologous genes into the vaccinia virus thymidine kinase (TK) gene during a single recombination event. This vector, pDAVAC2, contains bidirectional vaccinia P7.5 early/late promoter elements and two unique cloning sites. cDNA clones containing the complete coding sequences for the Lassa virus (Josiah strain) nucleoprotein (N) and glycoprotein (GPC) genes were inserted into the vaccinia TK gene using this transfer vector. The recombinant virus, V-LSGN-II, expressed proteins in cell culture that appeared to be authentic with respect to electrophoretic mobility, glycosylation, and post-translational cleavage. Indirect immunofluorescence (IFA) of recombinant virus-infected cells demonstrated both the bright granular and diffuse patterns of staining characteristic of the Lassa nucleoprotein and glycoprotein, respectively. Electron-dense inclusion bodies typical of arenavirus-infected cells were observed by electron microscopy in V-LSN and V-LSGN-II-infected cells, but not in V-LSGPC-infected cells. Mice inoculated with V-LSGN-II by intraperitoneal injection developed serum antibodies that reacted with authentic Lassa proteins in immunofluorescence and radioimmune precipitation assays. This recombinant virus represents an additional candidate for a Lassa fever vaccine and demonstrates the feasibility of expressing any two genes of interest in a single recombinant vaccinia virus through the use of the transfer vector pDAVAC2.

Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus.

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.

The nucleoprotein gene of Ebola virus: cloning, sequencing, and in vitro expression.

Genomic and messenger RNAs of a Zaire strain of Ebola virus were cloned, and inserts specific for the nucleoprotein gene were isolated and sequenced. The nucleoprotein gene is located proximal to the 3' end of the genome and is preceeded by a putative leader sequence. The gene begins with the transcriptional start site sequence 3'-UACUCCUUCUAAUU..., and ends with the polyadenylation site sequence 3'-... UAAUUCUUUUUU. The predicted coding region is 2217 bases in length and encodes a protein that contains 739 amino acids, with a calculated molecular weight of 83.3 kDa. The protein has an approximate net charge of -30 and can be divided into a hydrophobic N-terminal half and a hydrophilic and highly acidic C-terminal half. An in vitro transcript, generated from plasmid DNA containing the entire coding region, directs the synthesis of authentic nucleoprotein in a rabbit reticulocyte lysate system. The genomic organization and transcriptional signals of Ebola are similar to those of other nonsegmented, negative-strand RNA viruses, but nucleic acid or amino acid sequence comparisons indicate a lack of similarity.

Nucleotide sequence of the Lassa virus (Josiah strain) S genome RNA and amino acid sequence comparison of the N and GPC proteins to other arenaviruses.

The complete nucleotide sequence of the S genome RNA of the Josiah strain of Lassa virus was determined from cloned cDNA. The S RNA is 3402 nucleotides long with a calculated molecular weight of 1.09 x 10(6) Da. The nucleotide base composition is 26.84% adenine, 21.40% guanine, 22.75% cytosine, and 29.01% uridine. The 5' and 3' terminal nucleotide sequences are conserved and complimentary for 19 nucleotides, the nucleoprotein and glycoprotein genes are arranged in ambisense coding strategy, and the intergenic region contains an inverted complimentary sequence, as do all other arenavirus S RNAs characterized to date. Amino acid sequence comparisons between the nucleoproteins and glycoproteins of the Josiah and Nigerian (N sequences only) strains of Lassa virus, the WE and ARM strains of lymphocytic choriomeningitis virus (LCMV), Tacaribe, and Pichinde viruses are presented. These findings reveal that the G2 envelope glycoprotein is more conserved among different arenaviruses than the internal nucleoprotein.

Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection.

A cloned cDNA (1.65 kb) containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus. The Lassa virus glycoprotein precursor, GPC, and the posttranslational cleavage products, G1 and G2, were shown by Western blot analysis to be properly expressed in cells infected with the recombinant virus. Northern blot hybridization of total cytoplasmic RNA extracted from recombinant virus infected cells demonstrated the presence of RNA transcripts of appropriate size considering the site of transcription initiation from the vaccinia P7.5 promoter, the size of the Lassa glycoprotein gene, and the presumed location of the transcription terminator in the vaccinia thymidine kinase gene. All guinea pigs vaccinated with the recombinant virus survived a lethal challenge infection with Lassa virus, whereas 80% of control animals died. The vaccinated guinea pigs did, however, develop transient, low-grade, fevers and detectable viremias following infection with Lassa virus, indicating that protection was not complete.

Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA.

Two overlapping cDNA clones corresponding to the 5' region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an ambisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5' terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3' termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression.

Sequencing studies of pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA.

Analyses of the complete sequence of the 1.1 X 10(6)-dalton, small (S) RNA of the arenavirus Pichinde and virus-induced cellular RNA species have revealed that the viral nucleoprotein, N, is coded in a subgenomic, non-polyadenylated, virus-complementary mRNA corresponding to the 3' half of the viral RNA (Auperin et al., Virology 134:208-219, 1984). By contrast, a second S-coded product, presumably the viral glycoprotein precursor (GPC), is coded in a subgenomic, virus-sense mRNA corresponding to the 5' half of the RNA. Between the two genes is a unique RNA sequence that can be arranged in a hairpin configuration and may function as a transcription terminator for both genes. The term ambisense RNA is coined to describe this novel coding strategy of a viral RNA. The unique feature of the strategy is that the presumptive GPC mRNA and its translation product cannot be made until viral RNA replication has commenced. In addition, it allows the two subgenomic mRNA species to be regulated independently from each other or from other viral mRNA species. The implications of this strategy on possible mechanisms for the induction and maintenance of viral persistence, an important attribute of arenavirus infections, are discussed.

The sequences of the N protein gene and intergenic region of the S RNA of pichinde arenavirus.

Two overlapping DNA clones representing more than half of the Pichinde arenavirus S RNA segment were cloned into pBR322 and their nucleotide sequences were determined. The analyses predict that the viral nucleocapsid protein (N) is encoded in a reading frame in the viral complementary RNA sequence starting at viral S RNA nucleotide residue 84 from the 3' end and terminating with an opal codon at residues 1767-1769. The position of the termination codon has been confirmed by primer directed dideoxynucleotide sequencing. The N protein has a calculated size of 62,911 Da and a net positive charge of +9. Viral complementary 15 S mRNA that directs the synthesis of N protein and hybridizes to the predicted N gene DNA has been identified in infected cell extracts. A second nonoverlapping reading frame in the viral complementary sequence originates at nucleotide position 1827 and remains open for at least 71 amino acids (i.e., the extent of the second clone). A long stretch of hydrophobic amino acids is near the amino terminus of this predicted gene product. Between the two reading frames is a 60-nucleotide-long noncoding intergenic region. This nucleotide sequence can be arranged in hairpin configuration involving 14 G-C and 4 A-U base pairs. The possible function of this intergenic region in the regulation of transcription and/or translation is discussed.

Nucleotide sequence analyses and predicted coding of bunyavirus genome RNA species.

We performed 3' RNA sequence analyses of [(32)P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3'-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3'-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3'-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3'-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.