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The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes.

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.

Partial nucleotide sequences of the haemagglutinin (H) gene of 18 measles viruses isolated in the Gambia during 1994 and 1995 show a high degree of homology (> 98.5%) when compared with each other. These sequences form a cluster distinct from measles viruses isolated from other geographic regions and from the sequences of vaccine strains and two isolates from the Gambia from earlier years. Despite the low level of genetic heterogeneity observed, a common feature of the Gambian isolates is the presence of three nucleotide changes (at positions 7963, 8649 and 8653) not observed in isolates from other locations.

The sequences of the genes encoding the putative attachment (G) proteins of pathogenic (strain J3666) mouse lung-passaged and nonpathogenic (strain 15) tissue culture-passaged strains of pneumonia virus of mice (PVM) have been determined. In both cases the major polypeptide was synthesised from the second open reading frame (ORF), a feature also found in the G gene of respiratory syncytial (RS) virus, another pneumovirus. However, the ORFs of the G genes of the two PVM strains were initiated at different nucleotide positions in the mRNA and comparison of hydrophobicity profiles revealed the presence of the putative amino-terminal cytoplasmic domain in the strain J3666 G protein and its absence in the predicted G protein of PVM strain 15. In common with the G protein of RS virus, the gene product of both PVM strains contained a high serine, threonine, and proline content. Indirect immunofluorescence analysis of BSC-1 cells expressing the G gene products confirmed the surface location of the proteins. Thus, the absence of a cytoplasmic domain does not interfere with the translocation of the G protein of PVM strain 15. In vitro translation of mRNA from the two PVM genes directed the synthesis of a larger polypeptide with the G gene of PVM strain J3666 than was seen with strain 15 G gene. In addition, a second protein was seen with strain J3666 mRNA which was the same size as the strain 15 G protein.

Six different genotypes (designated lineages SHL1-6) of human respiratory syncytial (RS) virus have been defined by partial nucleotide sequence analysis of the variable SH and the hypervariable G membrane protein genes, and by restriction fragment analysis of the conserved N protein gene of viruses isolated in south Birmingham. Viruses of very similar genotype appear to be present worldwide at the present time. We have determined the nucleotide sequences of the fusion protein genes of five viruses isolated in south Birmingham in the same year, but belonging to different lineages, and have compared them with the sequences of four subgroup A viruses isolated at earlier times from diverse localities. The sequence diversity of the F genes of these five viruses, as measured by nucleotide (94.5-98.5%) and inferred amino acid (97.0-99.3%) identifies, is comparable with that of the nine subgroup A viruses considered as a whole. No sequence changes occur in any of the sites of known epitopes. Comparison of the nine subgroup A sequences with the published sequences of a subgroup B strain and three bovine RS viruses confirms that the F protein sequences are most divergent in the F2 region.

The variability of respiratory syncytial virus isolates from five successive epidemics in an urban population was determined. A total of 187 isolates of respiratory syncytial virus from the southern part of Birmingham, United Kingdom, were classified into subgroups A and B and were then further assigned to genetic lineages. Allocation of isolates into lineages was achieved by reverse transcription of infected cell RNA and then PCR amplification of selected regions of the genome; PCR products were examined by restriction mapping or nucleotide sequencing of parts of the nucleoprotein gene, the small hydrophobic protein gene, and the attachment protein gene. Previous work has shown that estimations of genetic diversity by analysis of genes coding for proteins likely (attachment protein) and unlikely (nucleoprotein and small hydrophobic protein) to be under immune pressure gave concordant results. Six genetic lineages of subgroup A isolates have been defined by this procedure; these isolates differ by up to 20% in the amino acid sequences of their attachment proteins; likewise, subgroup B isolates can be divided into two categories by restriction mapping of parts of their nucleoprotein and attachment protein genes. The same genetic lineages appeared to be present worldwide during the same period. The analysis of isolates from successive epidemics showed that different lineages predominated in each epidemic and that not all lineages were present in every epidemic. Some lineages appeared to increase in numbers over several years and then decline, possibly indicating a buildup of resistance in the community to a particular genotype.

Sequence analysis of a large number of clones derived from the carboxy-terminal one-third of the attachment (G) protein gene of subgroup A respiratory syncytial viruses revealed a region very prone to polymerase errors which resulted mainly in frameshifts because of the insertion or deletion of adenosine residues in some but not all runs of such residues. Such mutations were detected in 14% of clones derived from mRNA, 58% of clones derived from genomic-sense RNA, and 50% of clones derived from in vitro-transcribed RNA. This phenomenon appears to be dependent on the template sequence.

The nucleotide and deduced amino acid sequences of three genes of turkey rhinotracheitis virus (TRTV) together with the nucleotide sequences of the relevant intergenic regions were determined. The deduced amino acid sequence of one of the genes shows significant identity (42%) to that of the 22K protein of human respiratory syncytial virus (RSV). The TRTV 22K gene, like that of RSV, has a second open reading frame, although the amino acid sequence deduced from this reading frame does not show any similarity to the equivalent predicted RSV protein. The other two genes and their deduced amino acid sequences do not show any sequence similarity to the genes of other pneumoviruses. However, the hydrophobicity profiles of the predicted proteins do show similarities to those of the small hydrophobic (SH) and attachment protein (G) genes of RSV. The TRTV G gene is 1193 nucleotides in length and encodes a protein of 391 amino acids (M(r) 42984), which is rather larger than the RSV G protein (predicted M(r) 36000). The TRTV SH gene is 589 nucleotides in length, encoding a protein of 174 amino acids (M(r) 18797), which is considerably larger than the size of the RSV SH protein (M(r) 7500). The sequences of the intergenic regions derived from clones of polycistronic mRNAs and polymerase chain reaction products obtained with primers from different genes reveal the order on the virus genome to be 3' F-22K-SH-G 5'. This differs from the gene order of paramyxoviruses and morbilliviruses, which lack a 22K gene (and in some cases a SH gene), and the pneumoviruses RSV and pneumonia virus of mice, which have the F and 22K genes located after the G gene.

The gene encoding the fusion (F) glycoprotein of pneumonia virus of mice consists of 1657 bases and contains an open reading frame encoding 537 amino acids which is more similar to the F proteins of pneumoviruses than to those of other paramyxoviruses. Computer-assisted sequence analyses can be combined with data on the antigenicity of various F proteins to suggest a possible arrangement of secondary structure elements common to all pneumovirus and paramyxovirus F proteins.

The mutation responsible for the temperature-sensitive (ts) phenotype of mutant tsN19 (complementation group E) of respiratory syncytial virus has been located to the P protein gene. Viral protein synthesis was completely restricted at 39 degrees C, and the tsN19 P protein did not react with an anti-P monoclonal antibody (MAb) (3-5) at 33 degrees C. Reversion of temperature sensitivity restored reactivity with MAb 3-5. Nucleotide sequence determination and in vitro expression of cDNA clones of P mRNA derived from wild-type, tsN19 and non-ts revertant-infected cells, revealed that temperature sensitivity and loss of reactivity with MAb 3-5 were consequences of a Gly----Ser amino acid change at position 172. A low M(r) polypeptide, which represented the C-terminal 93 amino acids of the P protein, was produced by internal initiation in the P open reading frame during in vitro translation, and a similar product was detected transiently in vivo.

A consensus sequence for the P protein gene of the RSN-2 strain of respiratory syncytial (RS) virus was obtained by PCR amplification of cDNA obtained by reverse transcription. This established that the extent of sequence variation between two P genes of strains of antigenic subtype B is similar to that among A strains, confirming the conservation of P genes within subtypes and the divergence of the two antigenic subtypes of RS virus. The P protein of RS virus exhibits anomalous electrophoretic mobility with respect to its molecular weight. In vitro transcription and translation of RSN-2 strain cDNA possessing single point mutations revealed that substitutions involving charged amino acids in the carboxy-terminal region had a marked effect on the electrophoretic mobility of the P protein.

We have cloned eight intergenic regions from the Pneumovirus pneumonia virus of mice that link the nine small and medium sized genes previously described (Chambers et al., 1990). The nucleotide sequences of the clones confirm the locations of these genes and their mRNA transcripts in the viral genome. The intergenic regions vary in size from 2-56 nucleotides and show only faint homology to each other or to their analogues in respiratory syncytial virus. Sequence alignments suggest that the location of the transcriptional start site for the mRNA encoding the major nucleocapsid protein of pneumonia virus of mice and respiratory syncytial virus may have altered during virus evolution by gain or loss of a transcriptional start signal.

A simple and rapid procedure for determining the genotypes of viruses has been applied to analysis of genome subunit reassortment in heterologous crosses of Batai virus, Bunyamwera virus, and Maguari virus, three members of the Bunyamwera serogroup of bunyaviruses. The procedure for determining genotype made use of specific molecular probes to identify the parental origin of the L and M RNA subunits. Complementary DNA copies of the three RNA segments of Bunyamwera virus were prepared by reverse transcription using synthetic oligonucleotide primers for first and second strand synthesis. The cDNA transcripts were inserted into a pBR322 vector and gene-specific probes prepared from nick-translated plasmid DNA. L and M gene-specific probes were identified which could unequivocally discriminate Bunyamwera virus genome subunits in a dot-hybridization test using cytoplasmic RNA extracts immobilised on nitrocellulose filters. None of the S gene-specific probes were sufficiently discriminatory for use in this test. Instead the parental origin of the S RNA subunit was inferred from the electrophoretic mobility of the virion N protein. It was observed that reassortment did not occur at random in heterologous crosses of is mutants of the three viruses, and only the M RNA subunit appeared to segregate freely. However, unrestricted reassortment was observed when recombinant viruses with nonhomologous subunit combinations were used as the parental viruses. It was concluded, therefore, that restriction was mediated at the gene product level and that nonrandom reassortment was not due to incompatibility of genome subunits.

A simple method of the so-called pinpoint hybridization for the detection of genome RNAs and individual fragments of genome of the Bunyamwera family viruses is described. The method established linking of 2 out of 3 fragments of genome RNA of different members of the family.

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.