SEARCH · PubMed Health

Results for “genome segmentation”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

PubMed1995

Differentiation of cognate dsRNA genome segments of bluetongue virus reassortants by temperature gradient gel electrophoresis.

The analysis of reassortant viruses has been a valuable tool in the investigation of protein interaction and function in double-stranded (ds) RNA virus research. The differentiation of cognate dsRNA genome segments of reassortants is conventionally achieved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). However, due to a high degree of sequence homology among different bluetongue virus (BTV) serotypes, it is not uncommon to find that certain cognate dsRNA segments cannot be differentiated by SDS-PAGE. Temperature gradient gel electrophoresis (TGGE) has been shown to be a much more sensitive method of differentiating RNA or DNA fragments of high sequence homology. Here we report the preliminary application of TGGE in analysis of genomic reassortants of two BTV serotypes, 1 and 23. While six out of ten genome segments between BTV-1 and BTV-23 could not be resolved by SDS-PAGE, all of them were differentiated by TGGE. The ability of TGGE to distinguish between dsRNA segments of high sequence homology may also make it useful in the search for BTV genes responsible for defined characteristics, such as virulence, by differentiating wild-type and mutated gene segments of viruses displaying altered phenotypes.

Bluetongue virus↗

PubMed1991

Nucleotide sequences of genome segments S8, encoding a capsid protein, and S10, encoding a 36K protein, of rice gall dwarf virus.

The nucleotide sequences of DNAs complementary to the eighth (S8) and the tenth (S10) largest of the 12 genome segments of rice gall dwarf virus (RGDV) were determined. The S8 and S10 segments consist of 1578 and 1198 nucleotides, each with a single open reading frame extending for 1278 nucleotides from nucleotide 21, and 960 nucleotides from nucleotide 22, respectively. S8 encodes a polypeptide of 426 amino acids with an Mr of 47419. The amino acid sequences of several peptide fragments of the major outer capsid protein reported as 45K were contained in the predicted polypeptide. This protein, renamed the 47K protein, showed high homology with the outer capsid proteins of rice dwarf virus (RDV) and wound tumour virus (WTV); there was 56, 52 and 48% amino acid sequence identity between RGDV and WTV, RGDV and RDV, and RDV and WTV, respectively. S10 had the coding potential for a polypeptide of 320 amino acids with an Mr of 36,095 (36K protein), which exhibits 32% and 35% amino acid sequence identity with the predicted translation product of RDV S9 and the P9 capsid protein encoded by WTV S11, respectively. The conserved terminal sequences 5' GG...GAU 3' which are present in all genome segments of WTV and RDV so far analysed, and in S9 of RGDV, were also found in RGDV S8 and S10. This conserved sequence together with the segment-specific inverted repeats found in the terminal sequence of RGDV S8 and S10 are thus characteristic structures common to all three phytoreoviruses. The nucleotide sequence of the region surrounding the inverted repeats was more similar between RGDV and WTV than between RGDV and RDV.

Amino Acid Sequence↗

PubMed1992

The complete sequence of genome segment 8 of bluetongue virus, serotype 1, which encodes the nonstructural protein, NS2.

Bluetongue virus has a ten-segment double-stranded RNA genome, of which segment 8 encodes a nonstructural protein NS2. This protein is the only bluetongue viral protein to be phosphorylated and also has the ability to bind single-stranded RNA. At present, the function of NS2 is unknown and in order to analyse its characteristics in more detail, it was first necessary to obtain a full-length cDNA clone of the genome segment.

Amino Acid Sequence↗

PubMed1998

Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation.

A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3-4 kb genes of dsRNA viruses. The method of single primer amplification (Lambden et al., 1992) was adapted by the inclusion of a 3' poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro.

African Horse Sickness Virus↗

PubMed2002

Identification and characterisation of the genomic segment 7 of the infectious salmon anaemia virus genome.

The isolation and characterisation of a gene encoding the putative matrix proteins of infectious salmon anaemia virus (ISAV) is reported. Following identification of an ISAV-specific sequence from a cDNA library, RACE-PCR was used to identify a mRNA transcript of approximately 1.2 kb containing the ISAV consensus sequence GCTAAGA at the 5' end. Although the cDNA transcript and its putative protein product did not possess high homology with other orthomyxoviral sequences, similarity to a paramyxovirus fusion glycoprotein and viral cell surface proteins was identified. The size of this transcript suggested that it was derived from segment 7 of the ISAV genome and encoded the matrix proteins. Like syntenic segments of other orthomyxoviruses, this segment was shown to encode at least two matrix proteins, M1 and M2. The existence of M1 and M2 ISAV mRNA was demonstrated by RT-PCR and sequencing, with the M1 transcript being more abundant than the M2 in infected cell cultures, as is found in other orthomyxoviruses. Nucleotide sequence comparison of segment 7 of the ISAV genome from isolates of different geographic origin indicated it to be the one of the most variable of the ISAV genes characterised to date.

Amino Acid Sequence↗

PubMed1980

Comparison of human rotaviruses isolated in Mexico City and in Santiago, Chile, by electrophoretic migration of their double-stranded ribonucleic acid genome segments.

During the period October to December 1979, rotaviruses were obtained from infants and young children hospitalized with acute gastroenteritis in Mexico City and were compared by analysis of the migration of their double-stranded ribonucleic acid (RNA) genome segments in gel electrophoresis. Comparison of the results of this analysis and of those of similar studies carried out in 1977 and 1978 showed that the two rotavirus electropherotypes designated 2s and 21 have been continuously present and that the proportion in which these two types have been found in hospitalized patients has varied greatly year to year. The RNAs from rotaviruses 2s and 21 differed in the electrophoretic migraton of at least eight genome segments. However, RNAs from virus assigned to the same electrophoreotypes were not necessarily identical: on the basis of small but significant differences in the migration of segment 7, 8, or 9, isolates of types 2s and 21 could be assigned to two and three different subtypes, respectively. Human rotaviruses obtained in a distant geographical region, Santiago, Chile, in July 1979 had RNA electrophoretic patterns similar to that of electropherotype 21 but different from it in the migration of one or two of the larger RNA segments.

Child, Preschool↗

PubMed1989

The sequences of the S2 genome segments of reovirus serotype 3 and of the dsRNA-negative mutant ts447.

The most temperature-sensitive dsRNA-negative mutant of reovirus serotype 3 is ts447; the amount of dsRNA formed in cells infected with it at 39 degrees is less than 0.1% of that formed in cells with wt virus at 37 degrees. The genome segment in which this mutation is located is S2. We compare here the sequence of the S2 genome segment of wt reovirus serotype 3 with that of mutant ts447. The two sequences differ in three locations, at two of which there are C to U transitions, while at the third there is an A to G transition. All cause amino acid changes (Ala to Val, Ala to Val, and Asn to Asp, respectively). One mutation (at nucleotide position 581, which causes an Ala to Val change) causes the length of an alpha-helix to be significantly reduced and may be that which is responsible for the ts phenotype.

Amino Acid Sequence↗

PubMed1987

Assignment of the genome segment coding for the neutralizing epitope(s) of orbiviruses in the Great Island subgroup (Kemerovo serogroup).

Reassortant viruses were produced from high-frequency recombination events between temperature-sensitive (ts) mutants of Broadhaven (BRD) (S. R. Moss and P. A. Nuttall, Virus Res. 4, 331-336, 1986) and Wexford (WEX) viruses, two serotypes in the Kemerovo serogroup of orbiviruses. The parental origin of each of the 10 genomic segments comprising each reassortant was determined by polyacrylamide gel electrophoresis. Comparison of neutralization titers with the results of genomic dsRNA analyses revealed that genome segment 5 codes for the neutralizing epitope(s). Kemerovo group viruses therefore differ from orbiviruses of the bluetongue serogroup in which segment 2 codes for the neutralizing epitopes (M. J. Grubman, J. A. Appleton, and G. J. Letchworth, Virology 131, 355-366, 1983; J. Kahlon, K. Sugiyama, and P. Roy, J. Virol. 48, 627-632, 1983). Results also indicated that the ts lesions in mutants of recombination groups II, III, and VI were in segments 1, 5, and 4, respectively. Reassortment of segments 2 and 10 appeared to be nonrandom, and evidence of possible "linkage" was obtained for segments 3 and 9.

Animals↗

PubMed2004

Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus.

The ORF of genome segment 6 (S6) of rice ragged stunt oryzavirus (RRSV) Philippines isolate was cloned and sequenced based on the S6 sequence of the Thailand isolate. Pns6, the 71 kD product of S6 expressed in E. coli, was demonstrated to be a viral non-structural protein of RRSV by Western blotting. The gel mobility shift assays showed that Pns6 had nucleic acid binding activity. Pns6 could interact with single- and double-stranded forms of DNA and RNA, showing a preference for single-stranded nucleic acid and a slight preference for RRSV ssRNA over the rice ssRNA, as demonstrated by both competition and displacement assays. The binding of Pns6 to nucleic acids is strong and sequence non-specific. By using five truncated derivatives of Pns6, it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleic acid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plants and subsequent Western blotting had shown that Pns6 accumulated predominately in the cytoplasmic membrane fraction. The possible role of RRSV Pns6 in virus replication and assembly is discussed.

Amino Acid Sequence↗

PubMed1986

Coding strategy of the S genome segment of Hantaan virus.

Hantaan virus is the type species of the recently recognized Hantavirus genus of Bunyaviridae. The small (S) RNA segment of the negative-sense, tripartite genome was molecularly cloned and the nucleotide sequence was determined. The RNA sequence derived from the cDNA copy was found to contain 1696 nucleotides. A single open reading frame of sufficient size to encode the virus nucleocapsid protein was detected in the cDNA corresponding to viral complementary-sense RNA. RNA transcripts of the cDNA were synthesized with SP6 polymerase and were used to program cell-free reticulocyte lysate translation systems. Viral complementary-sense transcripts served as efficient messages in translation systems and generated Hantaan nucleocapsid protein. No translation products were detected when lysates were programmed with viral-sense transcripts. This coding assignment of the nucleocapsid protein to the viral complementary-sense RNA of the S genome segment is consistent with those of other members of this family. Unlike other Bunyaviridae, which encode both a nucleocapsid protein and a nonstructural (NSs) protein of similar sizes, a NSs protein has not been identified for Hantaan virus. Furthermore, other than the nucleocapsid protein gene sequence, the only potential open reading frame in Hantaan S RNA encoded a short, 48-amino acid polypeptide which initiated two codons beyond the termination of the nucleocapsid protein in the same reading frame. These data demonstrate that the coding strategy of the Hantaan virus S RNA is different than those reported for other viruses in this family.

Amino Acid Sequence↗

PubMed1983

A genetic map of reovirus: assignment of the newly defined mutant groups H, I, and J to genome segments.

Mutants representing three previously undefined reovirus type 3 mutant groups have been isolated following backcross of suppressed pseudorevertants to wild type (R.F. Ramig and B.N. Fields, 1979, Virology 92, 155-167; R. Ahmed, P.R. Chakraborty, A.F. Graham, R.F. Ramig, and B.N. Fields, 1980, J. Virol. 34, 383-389). The prototype mutant of each of the three new mutant groups was mapped by analysis of genome segment segregation in intertypic recombinants derived from crosses between the type 3 ts mutants and ts mutants of type 1 or type 2. Segregation analysis revealed the location of the group H prototype mutant tsH(26/8) to be genome segment M1, that of the group I prototype mutant tsI(138) to be segment L3, and that of the group J prototype mutant tsJ(128) to be segment S1. Mapping of the group I and J lesions required the identification of suppressed ts lesions in some of the intertypic rcombinant clones.

Animals↗

PubMed1990

Rotavirus genome segment 4 determines viral replication phenotype in cultured liver cells (HepG2).

One-step growth determinations were performed with five strains of rotavirus in HepG2, a cell line derived from human liver. Three virus strains (SA11-C13, SA11-C14, and RRV) replicated in HepG2 cells and attained yields 10- to 100-fold above input titers. Two virus strains (B223 and SA11-4F) failed to replicate above input titer. Analysis of reassortants that segregated the genes of parental virus pairs able and unable to replicate revealed that the HepG2 cell growth phenotype segregated with genome segment 4. Immunofluorescence analysis of infected HepG2 cells showed that the production of detectable antigen correlated with the growth phenotype and also segregated with genome segment 4. Thus, we conclude that (i) some virus strains were capable of replication in cultured liver cells while other strains could not replicate under identical conditions and that (ii) the inability of some virus strains to replicate resulted from a segment 4-associated block in replication before protein synthesis. These results are discussed in terms of what is known of the functions of VP4.

Antigens, Viral↗

PubMed1983

Identification of bluetongue virus type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity.

Double-stranded (ds) RNA was isolated from either partially purified bluetongue virus (BTV) type 17 or BTV-17 infected cells. The RNA was denatured with either methylmercury hydroxide or dimethyl sulfoxide and translated in a reticulocyte cell-free system. The translation products were found to be similar to BTV-17 specific polypeptides in infected cells when analyzed by polyacrylamide gel electrophoresis and immunoprecipitation with BTV-17 specific polyclonal mouse ascitic fluid. The 10 dsRNA BTV-17 genome segments were resolved by preparative gel electrophoresis and the coding assignment of each segment was determined by translation in a cell-free system. Immunoprecipitation of VP3 from infected cell lysates by a type-specific neutralizing monoclonal antibody identified this polypeptide, which is coded for by genome segment 2, as being involved in virus neutralization. Immunoprecipitation of in vitro translation products directed by total BTV-17 RNA with a monoclonal antibody that reacts with all the serotypes of BTV and some other orbiviruses and precipitates VP7 directly demonstrated that gene segment 8 codes for this intergroup-specific polypeptide.

Animals↗

PubMed1999

Precise packaging of the three genomic segments of the double-stranded-RNA bacteriophage phi6.

Bacteriophage phi6 has a genome of three segments of double-stranded RNA. Each virus particle contains one each of the three segments. Packaging is effected by the acquisition, in a serially dependent manner, of the plus strands of the genomic segments into empty procapsids. The empty procapsids are compressed in shape and expand during packaging. The packaging program involves discrete steps that are determined by the amount of RNA inside the procapsid. The steps involve the exposure and concealment of binding sites on the outer surface of the procapsid for the plus strands of the three genomic segments. The plus strand of segment S can be packaged alone, while packaging of the plus strand of segment M depends upon prior packaging of S. Packaging of the plus strand of L depends upon the prior packaging of M. Minus-strand synthesis begins when the particle has a full complement of plus strands. Plus-strand synthesis commences upon the completion of minus-strand synthesis. All of the reactions of packaging, minus-strand synthesis, and plus-strand synthesis can be accomplished in vitro with isolated procapsids. Live-virus constructions that are in accord with the model have been prepared. Mutant virus with changes in the packaging program have been isolated and analyzed.

Bacteriophage phi 6↗

PubMed2000

Characterization of phi8, a bacteriophage containing three double-stranded RNA genomic segments and distantly related to Phi6.

The three double-stranded RNA genomic segments of bacteriophage Phi8 were copied as cDNA, and their nucleotide sequences were determined. Although the organization of the genome is similar to that of Phi6, there is no similarity in either the nucleotide sequences or the amino acid sequences, with the exception of the motifs characteristic of viral RNA polymerases that are found in the presumptive polymerase sequence. Several features of the viral proteins differ markedly from those of Phi6. Although both phages are covered by a lipid-containing membrane, the protein compositions are very different. The most striking difference is that protein P8, which constitutes a shell around the procapsid in Phi6, is part of the membrane in Phi8. The host attachment protein consists of two peptides rather than one and the phage attaches directly to the lipopolysaccharide of the host rather than to a type IV pilus. The host range of Phi8 includes rough strains of Salmonella typhimurium and of pseudomonads

Amino Acid Motifs↗

PubMed2001

Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA.

Aquatic birnaviruses, such as infectious pancreatic necrosis virus (IPNV), cause serious diseases in a variety of fish species used worldwide in aquaculture and have also been isolated from a variety of healthy fish and shellfish species. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. To better understand genetic and biological diversity among these viruses, the nucleotide and deduced amino acid sequences were determined from cDNA of the large open reading frame (ORF) of genome segment A of the 9 type strains of Serogroup A and 4 other representative strains of Serotype A1, the predominant serotype in the United States. In addition, nucleotide and deduced amino acid sequences were determined for the VP2 coding region of a variety of isolates representing 5 of the 9 serotypes. VP2 is the major outer capsid protein of aquatic birnaviruses. RT-PCR was used to amplify a 2904 bp cDNA fragment including all but a few bp of the large ORF of genome segment A or a 1611 bp fragment representing the entire VP2 coding region. Nucleotide and deduced amino acid sequences were determined from the PCR products. Pairwise comparisons were made among our data and 2 other aquatic birnavirus sequences previously published. Several hypervariable regions were identified within the large ORF. The most divergent pair of viruses exhibited a similarity of 80.1% in the deduced amino acid sequence encoded by the large ORF. Genomic relationships revealed in a phylogenetic tree constructed from comparison of the deduced amino acid sequences of the large ORF demonstrated that these viruses were clustered into several genogroups. Phylogenetic comparison of the deduced amino acid sequences of the VP2 coding region of 28 aquatic birnavirus isolates, including the type strains of all 9 serotypes, demonstrated 6 genogroups, some of which were comprised of several genotypes. The most divergent pair of viruses exhibited a similarity of 81.2% in the deduced amino acid sequence from the VP2 coding region. In contrast to previous studies of much shorter genomic sequences within the C-terminus-pVP2/NS junction coding region, these genogroups based on the entire large ORF or the VP2 coding region generally correlated with geographical origin and serological classification. Isolates from the major Canadian serotypes were more closely related to the European isolates than to isolates from the United States.

Amino Acid Sequence↗

PubMed2004

In vivo excision and amplification of large human genomic segments using the Cre/loxP-and large T antigen/SV40 ori-mediated machinery.

In vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements. In this study, an in vivo excision and amplification system in human BJAB cells was devised by combining the Cre/loxP system of bacteriophage P1 and the large T antigen/SV40 ori system of Simian virus 40. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into 5'- and 3'-untranslated regions (UTRs) of the human iNOS. An SV40 ori sequence, which serves as a conditional replication system, was inserted between the loxP sites. Trans-acting genes cre and large T antigen, which were under the control of a tetracycline responsive promoter, were also inserted into the 5'- and 3'-UTRs of the iNOS, respectively, by homologous recombination. Upon induction by doxycycline, the 45-kb iNOS genomic fragment of human chromosome 17 flanked by two loxP sites was excised and amplified up to about 45 copies per cell. Our method is very useful for obtaining large genomic fragments in quantities directly from human cells without using foreign hosts. Therefore, our approach can be used effectively for gap sequencing of a genome, gene therapy, and functional analysis of unknown genes in human cells.

3' Untranslated Regions↗

PubMed2002

Rotavirus genome segment 7 (NSP3) is a determinant of extraintestinal spread in the neonatal mouse.

We used the neonatal mouse model of rotavirus infection to study extraintestinal spread following oral inoculation. Five-day-old pups were inoculated with either SA11-Cl3, SA11-Cl4, SA11-4F, RRV, or B223. By using virus detection in the liver as a proxy determination for extraintestinal spread, rotavirus strains capable of extraintestinal spread at high frequency (rhesus rotavirus [RRV]) and very low frequency (SA11-Cl4) were identified. Both strains productively infected the gastrointestinal tract. Oral inoculation of mice with RRV/ SA11-Cl4 reassortants and determination of virus titers in the gut and liver revealed that the extraintestinal spread phenotype segregated with RRV genome segment 7 to a high level of significance (P = 10(-3)). RRV segment 7 also segregated with the growth of virus in the gut (P = 10(-5)). Although infection of the gut was clearly required for tropism to the liver, there was no correlation between virus titers in the gut and detection of virus in the liver. Five days after intraperitoneal administration to bypass the gut barrier to virus spread, RRV and SA11-Cl4 both were recovered in the liver. However, only RRV was found in the liver following subcutaneous inoculation, suggesting that this peripheral site presented a similar barrier to virus spread as the gut. Sequence analysis of segment 7 from parental RRV and SA11-Cl4 and selected reassortants showed that (i) amino acid differences were distributed throughout the coding sequences and not concentrated in any particular functional motif and (ii) parental sequence was preserved in reassortants. These data support the hypothesis that NSP3, coded for by genome segment 7, plays a significant role in viral growth in the gut and spread to peripheral sites. The mechanism of NSP3-mediated tropism is under investigation.

Administration, Oral↗