Influenza B virus genome: complete nucleotide sequence of the influenza B/lee/40 virus genome RNA segment 5 encoding the nucleoprotein and comparison with the B/Singapore/222/79 nucleoprotein

The complete nucleotide sequence of a cloned full-length DNA copy of genome RNA segment 5 of influenza B/Lee/40 virus has been determined. The genome segment is 1841 nucleotides in length and is capable of coding for a nucleoprotein (NP) of 560 amino acids. Comparison with the only other known sequence of an influenza B virus nucleoprotein gene (B/Singapore/222/79) indicates striking homology. Only 113 nucleotide substitutions are present between the two strains in their protein coding region and these lead to only 22 amino acid substitutions between nucleoproteins of identical polypeptide chain length. Assuming a common lineage, this reflects a calculated rate of amino acid sequence divergence of 0.1% per year. Like its influenza A virus counterpart, the influenza B/Lee/40 nucleoprotein is a basic protein with a relatively even distribution of its charged residues. The remarkable conservation of nucleoprotein primary structure over a 39-year period probably reflects both selection for performance of specific functions and protection from antigenic selection by the host immune system.     

Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Uganda

The entire genomic RNA of the Gulu (Uganda 2000) strain of Ebola virus was sequenced and compared to the genomes of other filoviruses. This data represents the first comprehensive genetic analysis for a representative isolate of the Sudan species of Ebola virus. The genome organization of the Sudan species is nearly identical to that of the Zaire species, but the presence of a gene overlap (between GP and VP30 genes) and a longer trailer sequence distinguish it from that of the Reston species. As has been observed with other filoviruses, stemloop structures were predicted to form at the 5' end of Ebola Sudan mRNA molecules, and the genomic RNA termini showed a high degree of sequence complimentarity. Comparisons of the amino acid sequences of encoded gene products shows that there is a comparable level of identity or similarity between Ebola virus species, with Sudan and Zaire actually showing a slightly closer relationship to the Reston species than to one another. These comparisons also indicated that the VP24 is the most conserved Ebola virus protein (followed closely by the VP40 and L proteins), while the GP is the least conserved gene product. The most divergent regions were seen in the C-terminus of GP1 (mucin-like region) and within the C-terminal third of the nucleoprotein sequence.     

Sendai virus NP gene codes for a 524 amino acid NP protein

The complete nucleoprotein (NP) gene sequences of the Sendai virus Fushimi and 6/94 strains were determined. For both viruses an open reading frame of 524 amino acids can be predicted for the NP proteins. By comparing the sequences with others reported in the literature, the 5 noncoding region and the middle third of the coding region were found to be highly conserved. The carboxyl terminal part carries nine amino acid changes and a completely different sequence of the carboxyl terminus with a seven amino acid extension. This carboxyl terminus of the Sendai virus NP protein was confirmed using tryptic peptide sequence analysis.     

Structural homologies between RNA gene segments 10 and 11 from UK bovine, simian SA11, and human Wa rotaviruses

The nucleotide sequences of gene segments 10 and 11 from UK bovine rotavirus have been determined. Gene 10 is 751 nucleotides long and contains a single long open reading frame capable of coding for a protein of 175 amino acids. When compared with the published data for gene 10 of the simian rotavirus SA11 and human Wa strains it was found to be more closely related to the SA11 structure (92% nucleotide sequence homology; 97% amino acid sequence homology) than to the human Wa structure (84% nucleotide, 86% amino acid sequence homology). All three strains have two potential N-glycosylation sites in the hydrophobic N terminus of the gene 10 protein. Gene 11 from UK bovine rotavirus is 667 nucleotides long with a single long open reading frame capable of coding for a protein of 198 amino acids. When compared with the published sequence of gene 11 from the human rotavirus Wa, the UK bovine rotavirus gene 11 was found to be one nucleotide longer in the 5'-noncoding region and three nucleotides longer in the coding region. The nucleotide sequence homology was 86%. The predicted proteins coded by segment 11 in UK and Wa rotaviruses are both rich in serine and threonine (23%) and very hydrophilic, but differ appreciably in amino acid sequence (83% homology).     

Sequence analysis of the Streptococcus mutans scrB gene.

The complete nucleotide sequence of the Streptococcus mutans GS-5 scrB gene coding for sucrose-6-phosphate hydrolase activity was determined. A potential ribosome-binding site as well as promoter sequences were identified upstream from the gene. The deduced amino acid sequence of the enzyme suggested a molecular weight of 51,750, which is similar to that estimated for the enzyme isolated from strain GS-5. The enzyme is slightly acidic, with a pI of 5.9, and is a relatively hydrophilic protein. The nucleotide and amino acid sequences of the enzyme showed significant homology with those of the sacA protein from Bacillus subtilis. In addition, a region of amino acid homology with the S. mutans fructosyltransferase and B. subtilis levansucrase proteins was also detected.     

Molecular cloning of GRA4, a Toxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies.

Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.     

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.     

Whole genome sequencing and characterization of a virulent Newcastle disease virus isolated from an outbreak in Sweden

In this study, the complete genome sequence of a Newcastle disease virus (NDV) isolate collected from an outbreak in 1995 in chickens was fully characterized and compared with other NDV sequences. The genome was found to be 15,192 nucleotides long and to consist of six genes in the order 3′-NP-P-M-F-HN-L-5′, similar to other avian paramyxoviruses type-I. However, a six-nucleotide insertion was observed in the 5′ non-coding regions of the nucleoprotein (NP) gene, a feature that is unique to some NDV isolates. The isolate shows the amino acid sequence ¹¹²RRQKRF¹¹⁷ at the cleavage site of the F protein, which is identical to a known motif for virulent pathotypes of NDV. The phylogenetic analysis of the coding region of the F gene indicated that this isolate belongs to genotype VI, more specifically to genotype VId, along with isolates from the other European countries (Denmark, Switzerland and Austria). The same genotype caused outbreaks in the Middle East and Greece in the late 1960s, and in Hungary, in the early 1980s, suggesting a common source for these outbreaks.     

Lineage replacement accompanying duplication and rapid fixation of an RNA element in the nsP3 gene in a species of alphavirus

A sequence of thirty-six nucleotides in the nsP3 gene of Ross River virus (RRV), coding for the amino acid sequence HADTVSLDSTVS, was duplicated some time between 1969 and 1979 coinciding with the appearance of a new lineage of this virus and with a major outbreak of Epidemic Polyarthritis among residents of the Pacific Islands. This lineage of RRV continues to circulate throughout Australia and both earlier lineages, which lacked the duplicated element, now are extinct. Multiple copies of several other elements also were observed in this region of the nsP3 gene in all lineages of RRV. Multiple copies of one of these, coding for the amino acid sequence P*P*PR, were detected in the C-terminal region of the nsP3 protein of all alphaviruses except those of African origin. The fixation of duplications and insertions in 3′ region of nsP3 genes from all lineages of alphaviruses, suggests they provide some fitness advantage.     

Screening for mutations in the exon 26 of the apolipoprotein B gene in hypercholesterolemic finnish families by the single-strand conformation polymorphism method

To date, the only known apolipoprotein B (apo B) mutation causing hypercholesteroletnia is the apo B 3500 Arg → Gln or the familial defective apo B (FDB) mutation. This mutation has not been detected in the Finnish population. We have set up a systematic single-strand conformation polymorphism (SSCP) analysis-based screening method to search for other mutations in the exon 26 of the apo B gene in 21 Finnish hypercholesterolemic probands. The 7572-bp exon 26 covers half of the coding region of the gene including the DNA sequence coding for the putative low-density lipoprotein (LDL) receptor binding site on the apo B protein. Exon 26 was amplified as six 1190- to 1435-bp fragments, each of which was further split into three smaller 213- to 579-bp segments by restriction enzymes. These digestion products were run on nondenaturing polyacrylamide gels using at least three different electrophoretic ccnditions and autoradiographed. All previously known genetic variants in the exon 26 were detected by the SSCP method. A C→T change at nucleotide 7064, in complete association with the XbaI site, was characterized by direct sequencing. This variant did not affect the amino acid sequence of the apo B protein. The SSCP-based procedure appears suitable for systematic screening for DNA sequence changes in large coding regions. © 1994 Wiley-Liss, Inc.     

Conservation of the 3' terminal nucleotide sequences of Ebola and Marburg virus

The 3' RNA base sequences of several Marburg (MBG) and Ebola (EBO) virus isolates have been determined. A comparison of these 3' terminal noncoding sequences with those of other negative strand RNA viruses suggests a unique phylogenic niche for Marburg and Ebola viruses. The translation initiation site and 35 N-terminal amino acids of the 3' proximal coding gene of a Zaire strain of Ebola virus was predicted. In addition, putative leader RNA sequences preceding the first gene are discussed in terms of possible regulatory functions.     

Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated.     

Evolutionary analyses of five US bluetongue viruses using the cognate S2 genes.

Full-length cDNA copies of S2 genes (segment 8), coding for non-structural protein 2 (NS2) of bluetongue virus serotypes 2, 11, 13 and 17 were selectively synthesized by a modified polymerase chain reaction (Clamp-R) and cloned into the PstI site of the pUC19 plasmid. Each of these S2 cognate genes was 1125 nucleotides in length with an initiation and a termination codon at nucleotides 20-22 and 1082-1084, respectively, resulting in a long open reading frame capable of coding for a protein of 354 amino acids. The deduced amino acid sequence of NS2 protein had a high concentration of lysine and contained a relatively low number of tryptophan and histidine residues. There was a highly conserved hydrophilic region at the carboxyl termini of predicted NS2 proteins in all five BTV serotypes, even though the amino acid sequence in this region in BTV-2 was more variable than in the other four serotypes. There was significant sequence homology of the cognate S2 genes at both the nucleotide and the amino acid levels. Phylogenetic analyses using the S2 gene sequences indicated that BTV-10, -11, -13 and -17 were more closely related and BTV-2 was the most distantly related serotype among the five US bluetongue viruses.