Viral Genome Segments Research Articles

Phylogenetic analysis of African horse sickness virus segment 10: sequence variation, virulence characteristics and cell exit.

African horse sickness virus (AHSV) genome segment 10 encodes the non-structural proteins NS3/NS3a, which is involved in release of virus from cells. Full length segment 10 cDNAs were amplified by reverse transcription-polymerase chain reaction, from isolates of AHSV serotypes 2, 3, 4, 5, 7, 8 and 9. These cDNAs were cloned, sequenced and their phylogenetic relationships analysed. High levels of sequence homology were detected in segment 10 from some isolates of different serotypes, confirming that they could be grouped on this basis (serotypes 4, 5, 6 and 9 (group alpha); serotypes 3 and 7 (group beta); serotypes 1, 2, and 8 (group gamma). However, data from bluetongue virus (the prototype orbivirus) indicate that the AHSV serotype is determined exclusively by the structural outer coat proteins VP2 and VP5, encoded by genome segments 2 and 5 respectively. Therefore, as a direct consequence of genome segment reassortment between AHSV strains from different serotypes, the differences observed in segment 10 do not give a reliable indication of virus serotype. Segment 10 of AHSV 3 (virulent) and AHSV 3att (attenuated) were also analysed. These strains, together with AHSV 8, have been used to study of the genetic basis of virulence using reassortment (O'Hara et al., this publication). Virus release studies, using Culicoides cell cultures, indicate that differences in segment 10 of AHSV 3att and 8 can influence the timing of virus release from the infected cell.

Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein lambda1.

A previously identified nucleoside triphosphatase activity in mammalian reovirus cores was further characterized by comparing two reovirus strains whose cores differ in their efficiencies of ATP hydrolysis. In assays using a panel of reassortant viruses derived from these strains, the difference in ATPase activity at standard conditions was genetically associated with viral genome segment L3, encoding protein lambda1, a major constituent of the core shell that possesses sequence motifs characteristic of other ATPases. The ATPase activity of cores was affected by several other reaction components, including temperature, pH, nature and concentration of monovalent and divalent cations, and nature and concentration of anions. A strain difference in the response of core ATPase activity to monovalent acetate salts was also mapped to L3/lambda1 by using reassortant viruses. Experiments with different nucleoside triphosphates demonstrated that ATP is the preferred ribonucleotide substrate for cores of both strains. Other experiments suggested that the ATPase is latent in reovirus virions and infectious subviral particles but undergoes activation during production of cores in close association with the protease-mediated degradation of outer-capsid protein mu1 and its cleavage products, suggesting that mu1 may play a role in regulating the ATPase.

Segmented Structure of Separate and Transposable DNA and RNA Elements as Suggested by Their Size Distributions

A collection of about 1000 different eukaryotic and prokaryotic DNA mobile and separate elements is compiled from literature—;transposons, plasmids, extrachromosomal circular DNA, insertion sequences, as well as viral genomes and separate genome segments. Only small elements are collected, upto 2000 base pairs. Analysis of the sequence length distributions of the elements reveals that certain sizes are clearly preferred, namely those which correspond to multiples of about 345 bp in eukaryotes and multiples of about 210 bp in prokaryotes. This provides additional evidence in support of the theory (1) that segmented structure is characteristic of not only protein-coding sequences (2) but rather of genomes in general. In particular, it confirms the prediction (1) that mobile and separate elements would also be segmented.

Modes of Seoul virus infections: persistency in newborn rats and transiency in adult rats.

To understand the mode of persistent infection of Seoul virus in rodents, we examined the distribution of the virus genome and antibody production in infected rats. When 1-day-old rats were inoculated with the KI-83-262 strain, the S segment of viral genome was detected in sera, clots, lungs and kidneys from 3 to 184 days post inoculation (d.p.i.) by nested reverse transcriptase PCR. On the other hand, when 7-week-old rats were infected with this virus, viral genome was detected only in the lungs from 3 to 50 d.p.i. The neutralizing antibody titers of rats inoculated at 1-day of age were higher than those of rats inoculated at 7 weeks of age. In both age groups, however, the IgG avidity of antibody increased along with the course of infection. We found that urban rats (Rattus norvegicus) infected early in life harbored the virus for more than 6 months.

Sequence Analysis of the Bicistronic Drosophila X Virus Genome Segment A and Its Encoded Polypeptides

Drosophila X virus represents the entomobirnavirus genus of the Family Birnaviridae. Segment A of this bisegmented dsRNA containing virus was cloned and sequenced. The 3360-bp-long nucleotide sequence revealed the presence of two open reading frames (ORFs). A large ORF of 3096 nucleotides, which is flanked by a 107-bp 5′ and a 157-bp 3′-untranslated region, and a 711-nucleotide-long small ORF located within the carboxy half of the large ORF but in a different reading frame. The large ORF encodes a 114-kDa polyprotein which is cotranslationally processed by the virus-coded protease VP4 to generate preVP2, VP3, and VP4 (VP1 is encoded by genome segment B). N-terminal amino acid sequencing of VP3 and VP4 established the order NH2-preVP2-VP4-VP3-COOH within the polyprotein. The small ORF straddles the VP4/VP3 junction and is capable of encoding a basic, arginine-rich 27-kDa polypeptide which so far has not been detected in infected cells. The amino acid sequences specified by the two ORFs were compared to those of infectious pancreatic necrosis virus (IPNV) and infectious bursal disease virus (IBDV) that represent the two other genera (aquabirnavirus and avibirnavirus) of the Birnaviridae family. Significant sequence homology among the three viruses was found to be restricted to the amino and carboxy regions of preVP2 and to a small 21-residue-long domain near the carboxy terminal region of VP3. Significant sequence homology is exhibited by the small ORFs of the three viruses.

Defective RNAs Inhibit the Assembly of Influenza Virus Genome Segments in a Segment-Specific Manner

Four avian influenza viruses have been generated, each containing a single extra defective RNA segment in addition to the eight standard segments. Three of the extra RNAs were derived from segment 1 and the fourth from segment 2. Chick embryo fibroblast cells were infected with each virus, and a wild-type virus. Virus RNA was quantified in extracts of virus-infected cells and in virus released by 10 hr postinfection using reverse transcription and by Northern blot analysis. In the case of two of the viruses the presence of the defective RNA did not markedly affect the accumulation of virus RNA within the infected cell, but significantly and selectively reduced the amount of the “parent” segment in released virus. This effect was reduced in a third virus. In a fourth virus, defective RNA was found to be present at a low-input multiplicity and results were varied. Mixed infections of one of the viruses with a closely related wild-type virus resulted in reduction of the corresponding vRNA segment of the nondefective virus. We conclude that assembly of influenza virus segments is not a purely random process.

Restriction fragment profiles of genome segment A of infectious bursal disease virus correlate with serotype and geographical origin of avibirnaviruses

Previous analysis of the two serotypes of infectious bursal disease virus (IBDV) have demonstrated the correlation between antigenicity and similarities of nucleotide and amino acid sequences of the VP2 coding region in genome segment A. Restriction fragment profiles of genomic segment A cDNA of five IBDV isolates (QC-2 and QT-1 of serotype 1, SK9, and Nos. 39 and 52 of serotype 2) were determined in order to establish the genetic relationship of these viruses to other avibirnaviruses. The restriction fragment profiles using three of seven restriction enzymes (SacI which cuts in the VP2 region, DraIII which cuts in the VP3 region, and EcoRI which cuts in the VP4 region) were used to place QC-2, QT-1, SK9, No. 39, and No. 52 within the phylogenetic tree among seven other avibirnaviruses of known sequence. The two IBDV serotypes corresponded to two genotypes on the basis of the presence or absence of the SacI restriction site. The serotype 1 cluster of strains was further differentiated into five minor clusters on the basis of the PstI, EcoRI, BamHI, HindIII, DraIII, and Bsu361 restriction sites, which emphasized the geographical origins of the strains. It is concluded that restriction analysis of cDNA of the whole viral genomic segment A allows differentiation of IBDV isolates on the basis of their antigenicity and geographical origin.

Topography and immunogenicity of bluetongue virus VP7 epitopes

The core of bluetongue virus (BTV) consists of ten dsRNA viral genome segments and five proteins, including two major (VP7 and VP3) and three minor (VP1, VP4 and VP6) components. The major core protein VP7 is believed to be an important structural constituent because it interacts, not only with the underlying core protein VP3, but also with two outer capsid proteins (VP2 and VP5). In this communication we summarise data on the mapping of at least six different epitopes of VP7 distributed along the molecule. Two of the six epitopes have not been mapped previously. The accessibility of these epitopes in intact virions and core particles was analysed using immunoelectron microscopy. The epitope located near the N-terminus of VP7 was accessible at the surface of intact virions and core particles. Epitopes in other parts of the VP7 molecule were detected weakly in core particles but not in intact virions. These results support the proposal that VP7 molecules are orientated with their N-terminus accessible on the surface of either the particle or at least one of the three different channels observed by cryoelectron microscopy in the outer capsid layer. Analysis of the immune response to BTV-infected or -immunised sheep and rabbits to three selected epitopes, which are located in different regions of the VP7 molecule, demonstrated that all of them were recognised by the animals tested. These results provided further molecular evidence suggesting that VP7 is indeed a major immunogenic antigen ideal for BTV antibody detection.

Naturally Occurring Sin Nombre Virus Genetic Reassortants

Genetic reassortment has been shown to play an important role in the evolution of several segmented RNA viruses and in the epidemiology of associated diseases. Sin Nombre (SN) virus is the cause of hantavirus pulmonary syndrome throughout the western United States. Like other hantaviruses, it possesses a genome consisting of three negative-sense RNA segments, S, M, and L. Recent analysis has demonstrated the presence of at least three different hantaviruses in Nevada and eastern California, including SN, Prospect Hill-like, and El Moro Canyon-like viruses. In addition, two distinct lineages of SN virus can be found inPeromyscus maniculatusrodents (sometimes in close proximity) trapped at study sites in this region. Data obtained by phylogenetic analysis of sequence differences detected among the S, M, and L genome segments of these SN viruses are consistent with reassortment having taken place between SN virus genetic variants. The results suggest that M (and to a lesser extent S or L) genome segment flow occurs within SN virus populations inP. maniculatusin this region. No reassortment was detected between SN virus and other hantavirus types present in the area. This finding suggests that as genetic distance increases, the frequency of formation of viable reassortants decreases, or that hantaviruses which are primarily maintained in different rodent hosts rarely have the opportunity to genetically interact.

Molecular analysis of rice ragged stunt oryzavirus segment 9 and sequence conservation among isolates from Thailand and India.

Nucleotide sequences of rice ragged stunt virus (RRSV) genome segment 9 (S9) from a Thai and an Indian isolate were determined. Both sequences are 1132 bp long, contain a single large open reading frame (ORF) spanning nucleotide residues 14 to 1027 and are capable of encoding a protein of 38.5K. The two isolates are 94.6% and 99.4% identical at the nucleotide and amino acid level, respectively. The authenticity of these coding sequences was confirmed by identifying a approximately 38K protein in the RRSV particle with an N-terminal amino acid sequence identical to that inferred from the S9 ORF. Furthermore, cDNA of S9 from each isolate incorporated into the bacterial expression vector pGEX3-X produced a fusion protein that reacted with antibodies raised against purified RRSV particles. Cleaving these fusion proteins with protease factor X liberated a approximately 38K polypeptide.

P-41 Cell transformation by transfection of hepatitis C virus genome segment encoding NS3

P-41 Cell transformation by transfection of hepatitis C virus genome segment encoding NS3

Rice ragged stunt Oryzavirus genome segment 9 encodes a 38 600 Mr structural protein.

The complete nucleotide sequence of rice ragged stunt virus genome segment 9 (S9) was determined. The S9 segment is 1132 nucleotides long and has a long open reading frame starting from the first AUG codon at nucleotide position 14-16 and terminating at a UAG codon located at 1028-1030, which could encode a polypeptide with an Mr of 38 600 (P9). The encoded polypeptide has no sequence homology to polypeptides of any other plant reoviruses published previously. An immunological study demonstrated that P9 was the smallest of the structural proteins. The P9 polypeptide was expressed as a fusion protein with maltose binding protein in Escherichia coli. Antisera to purified virions and to the fusion protein reacted with both the bacterially expressed polypeptide and the smallest polypeptide of the purified virus in immunoblotting analyses.

Assembly of double-stranded RNA viruses: bacteriophage ø6 and yeast virus L-A

Double-stranded RNA viruses have a virion-associated RNA-dependent RNA polymerase activity which is involved in such critical steps of viral assembly as genome packaging and minus strand synthesis. In vitro studies of a bacterial dsRNA virus, ø6, and a yeast virus, L-A, have shed light on capsid formation as well as on the protein/RNA interactions and packaging of the viral genomes. In the ø6 system, an empty dodecahedral polymerase complex (procapsid) composed of four protein species is formed without the help of other viral proteins or RNA. This particle packages positive sense viral RNA genome segments in an ATP dependent reaction. The presence of all rNTPs allows the synthesis of complementary (-) strands within the particle. Self-assembly of an additional protein shell (composed of protein P8) around this particle takes place in the presence of Ca2+ ions. In vivo, these nucleocapsids obtain an envelope while still residing in the cell cytoplasm. L-A, in contrast, is not known to make a prohead structure. The Pol domain of L-A's Gag-Pol fusion protein is necessary for packaging of the (+) strand RNA and probably actually binds to the (+) strand packaging site (a stem-loop with a protruding A) insuring its packaging while the Gag domain primes polymerization of the coat protein. N-Acetylation of Gag by the host MAK3 N-acetyltransferase is necessary for proper assembly, and the ratio of Gag-Pol/Gag, determined by the efficiency of - 1 ribosomal frameshifting, is critical for propagation of the M1 satellite dsRNA.

Expression and Sequence Analysis of Gene 7 of the IDIR Agent (Group B Rotavirus): Similarity with NS53 of Group A Rotavirus

The seventh genomic segment of the IDIR strain of group B rotavirus was cloned, sequenced, and expressed in vitro in order to evaluate the gene coding assignment by comparison with group A rotaviruses (GAR). Viral genomic RNA for these reactions was obtained directly from fecal specimens of infected infant rats. IDIR virus gene 7 (IDIRg7) contained 1276 bp. Both 5′ and 3′ termini resembled those reported for other IDIR virus genomic segments. Two open reading frames (ORF) were predicted from the gene sequence; a long ORF from bases 258-1217 and a short ORF from bases 41-385. The first AUG of the long ORF was flanked by sequences associated with highly efficient translation, but less efficient translation was predicted for the short ORF. In vitro transcription and translation of IDIRg7 demonstrated a product consistent with polypeptide synthesis from the long ORE but not the short ORF. Convalescent rat antibody directed against the IDIR agent specifically immunoprecipitated the IDIRg7 protein end indicated that the in vitro translation product was indeed encoded by the virus genome. Thus, the untranslated 5′ region of IDIRg7 was longer than that previously described for any major rotavirus product. Comparison of the IDIRg7 sequence indicated 51% similarity and 18% identity with the amino acid residues of NS53 of the SA11 strain of GAR. However, the sequence of IDIRg7 did not share the putative zinc binding region postulated for NS53 of rotavirus groups A and C. It will be of interest in future experiments to evaluate the function of the IDIRg7 product following large-scale synthesis by alternative expression systems such as vaccinia or baculovirus.

Epitope Mapping Studies with Neutralizing and Non-neutralizing Monoclonal Antibodies to the G1 and G2 Envelope Glycoproteins of Hantaan Virus

Epitopes recognized by three G1-specific and two G2-specific neutralizing monoclonal antibodies to Hantaan virus were mapped by sequence analyses of the complete M genome segments of neutralization escape variant viruses. For each variant, we detected nucleotide sequence substitutions which resulted in a single amino acid change in either the G1 or G2 protein. Serological properties of the variant viruses correlated with changes identified by nucleotide sequence analyses. To map epitopes recognized by three G1-specific and six G2-specific, non-neutralizing monoclonal antibodies, we prepared genes, truncated at the carboxy terminal coding regions of G1 or G2, and expressed them with baculovirus recombinants or transiently in a vaccinia/T7 RNA polymerase system. Reactivities of the monoclonal antibodies with the truncated proteins were monitored by immune precipitation of the radiolabeled, truncated glycoproteins. We determined that all three of the G1-specific antibodies reacted with truncated proteins, which retained the amino terminal one-third of G1, but lost reactivity with shorter G1 proteins. The G2-specific antibodies only recognized G2 proteins, which retained approximately 80% of the G2 gene.

Polydnavirus genome segment families in the ichneumonid parasitoid Hyposoter fugitivus.

Sequences homologous to encapsidated polydnavirus genome segments are routinely detected in parasitoid chromosomal DNA; typically, each viral genome segment hybridizes to a single cognate chromosomal locus. In the present study, we show that in some cases, two or more viral genome segments may hybridize to the same chromosomal locus. Genome segments of this type invariably share a majority of restriction enzyme sites, a fact suggesting derivation from a common template. Families of viral genome segments appear to be relatively common in the Hyposoter fugitivus polydnavirus genome.

Difference in Electrophoretic Pattern of the Rice Dwarf Virus Genome Segments in Agarose and Polyacrylamide Gel Electrophoresis.

Difference in Electrophoretic Pattern of the Rice Dwarf Virus Genome Segments in Agarose and Polyacrylamide Gel Electrophoresis.

Construction of a transducing virus from double-stranded RNA bacteriophage phi6: establishment of carrier states in host cells

Bacteriophage phi 6 contains three double-stranded RNA (dsRNA) genomic segments. We have constructed a plasmid that contains a cDNA copy of the middle (M) segment, with a gene for kanamycin resistance (kan) inserted into the PstI site. A transcript of this cDNA was incorporated in vitro into procapsids along with natural transcripts of the S and L segments. The procapsids were coated with nucleocapsid surface protein P8 and transfected into Pseudomonas syringae pv. phaseolicola. The resulting infectious virus, phi 6 K1, was found to contain an M segment that was 1.2 kbp larger than the normal 4.1 kbp. K1 formed small, turbid plaques, and its genome was unstable. Preparations of K1 contained from about 0.1 to 10% large, clear-plaque forms of the virus which were usually missing the kan gene, and in some cases, the resulting segment M was smaller than its normal size. Cells picked from lawns of host cells infected with K1 yielded colonies that were resistant to kanamycin (Kan). These colonies could be passaged on kanamycin-containing medium. The cells were found to contain large amounts of dsRNA corresponding to the viral genomic segments. Some strains continued to produce viable phage, while others lost this ability. One strain completely lost the small genomic segment S. Approximately 1 in 10,000 infected cells acquired the carrier state with the original phage isolate K1. However, we isolated a viral mutant that was able to induce the carrier state in 10 to 20% of the infected cells. The ability to use drug resistance as a test for the carrier state makes this system very useful for the study of the mechanisms of induction of persistent infections.

Evidence for a chromosomal location of polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus.

Evidence is presented in support of a chromosomal location for sequences homologous to polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus. In this study, four different viral genome segments were cloned and used as probes against genomic DNA extracted from male parasitoids and digested with a variety of restriction enzymes. Each probe typically identified a single off-size fragment (OSF) in the case of enzymes not cutting viral genome segments, while two OSFs were generated by enzymes cutting at one and two sites. While extra OSFs were occasionally observed, these were invariably found to be due to the presence of polymorphic restriction sites in flanking chromosomal DNA. Analysis of these data suggests that a single, stable chromosomal locus exists for sequences homologous to each viral genome segment; the data also indicate that viral and cognate parasitoid genomic DNAs are largely if not entirely colinear.

Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host.

The polydnavirus Campoletis sonorensis virus (CsV) is present in the oviducts of all adult C. sonorensis female wasps and appears to be required for these wasps to parasitize hosts successfully. Physical mapping, Southern blot analysis, and nucleotide sequence analysis demonstrate that the viral DNA B-specific sequences in cloned wasp DNA are colinear with viral genomic segment DNA B from nucleocapsids and are covalently linked to nonviral wasp sequences. Integrated DNA B terminates in 59-nucleotide imperfect direct repeats, but a single repeat exists in the extrachromosomal superhelical viral DNA B. Sequences near each junction form imperfect inverted repeats with sequences near the ends of an internal viral 540-base-pair repeat element gene. CsV appears to be the first documented integrated, nonretroviral DNA virus of insects and probably is vertically transmitted as a provirus.