Determination of the genome composition of influenza virus reassortants using multiplex reverse transcription-polymerase chain reaction followed by fluorescent single-strand conformation polymorphism analysis.

The influenza virus genome is composed of eight negative-strand RNA segments. In cells coinfected with two or more influenza strains, the genomic RNAs reassort at random, resulting in progeny viruses (reassortants) that contain genes derived from each parent. Genetic reassortment among influenza viruses occurs naturally and plays an important role in viral epidemiology and pathogenicity. The reassortment process is also utilized for the annual production of influenza vaccines. Each year, the two gene segments that encode the major surface antigens of the current virulent, wild-type viruses are reassorted with the remaining six gene segments of a laboratory-derived vaccine or "master donor" strain. As the gene reassortment appears to be random, identifying a progeny virus with the desired gene constellation can be labor-intensive. We developed a streamlined, cost-effective method to genotype influenza viruses that combines multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent single-strand conformation polymorphism (SSCP) analysis. This method utilizes oligonucleotide primers labeled with one of three fluorescent dyes to generate RT-PCR products for each gene segment in a multiplex configuration. The RT-PCR products of the reassortants, wild-type, and master donor viruses are then electrophoresed under SSCP conditions. The viral origin of each gene segment can be identified by fluorescence and mobility shift patterns of the corresponding RT-PCR products. We demonstrate the utility of this method in differentiating the genes of a master donor strain, several wild-type viruses, and vaccine reassortants.

Rescue of a 7502-nucleotide (49.3% of full-length) synthetic analog of respiratory syncytial virus genomic RNA.

A cDNA was constructed to encode an internally-truncated version of negative-sense genomic RNA (vRNA) of respiratory syncytial virus (RSV) containing (in 3' to 5' order) the 3' vRNA leader region, the complement of the open reading frame for bacterial chloramphenicol acetyl transferase (CAT) under the control of RSV gene-start and gene-end signals, an intergenic nucleotide, the complete L gene including the gene-start and gene-end signals, and the 5' vRNA trailer region. The encoded vRNA analog (RSV-CAT-L) would be 7502 nucleotides (nt) in length, 49.3% of the complete 15,222-nt parental vRNA. RSV-CAT-L vRNA was synthesized in vitro from HgaI-digested cDNA and transfected into RSV-infected cells. The vRNA was "rescued" such that it was expressed to yield CAT and was packaged into particles that could be passaged to express CAT in fresh cells. The efficiency of rescue was greatly improved by a single nucleotide substitution in the leader region that had been found to increase the efficiency of rescue and passage of the previously described 935-nt RSV-CAT vRNA. Compared to the 8-fold smaller RSV-CAT vRNA, and adjusted to molar equivalence of transfected RNA, RSV-CAT-L vRNA was 119- to 347-fold less efficient in expressing CAT upon transfection, whereas RSV-CAT-L vRNA containing the above-mentioned nucleotide substitution was 15.8-fold less efficient.

Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.

Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene.

The viral genomic RNA (vRNA) of human respiratory syncytial virus is a nonsegmented negative strand that is not infectious alone. To develop methods for complementing synthetic vRNA with viral proteins, a cDNA was constructed to encode a vRNA in which all of the viral protein-coding sequences were removed and replaced with a negative-sense copy of the bacterial chloramphenicol acetyltransferase gene. Upon transfection into respiratory syncytial virus-infected cells, the synthetic vRNA was "rescued" such that it was amplified, expressed, and packaged into infectious virions. A heterologous paramyxovirus, parainfluenza virus 3, was inactive in rescue. Further internal deletions mapped the cis-acting viral sequences required for rescue to two segments totaling 105 nucleotides (nt) derived from the two vRNA ends. Rescue was unaffected by replacement of the 44-nt 3'-terminal leader region with a 50-nt sequence that is complementary to the 5' terminus and represents the 3' end of the positive-sense replicative intermediate RNA. This 5'-end complement was related to the parental leader region only near the 3' terminus (91% or 73% identical for the first 11 or 22 nt, respectively). The addition of 11 heterologous nt to the 3' end of the parental leader region ablated rescue, suggesting that the 3'-proximal conserved domain is required and cannot function from an internal site. However, deletion of the 3'-terminal 3 nt, or a double transition at positions 4 and 5, had no effect on rescue. Thus, the 3'-terminal 5 nt, although conserved between 3' ends of the negative- and positive-sense RNAs, do not appear to be essential.

Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants.

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.

Sequence analysis of the polymerase L gene of human respiratory syncytial virus and predicted phylogeny of nonsegmented negative-strand viruses.

The complete nucleotide sequence of the large (L) polymerase gene of human respiratory syncytial virus (RSV) strain A2 was determined by analysis of cloned-cDNAs representing the entire gene and confirmed in part by dideoxy sequencing of genomic RNA. The RSV L gene is 6578 nucleotides in length and contains a single major open reading frame that encodes a protein of 2165 amino acids. The molecular weight (250,226) and amino acid composition of the deduced RSV L protein are similar to those of other negative-strand RNA viruses. Regions of statistically significant amino acid sequence similarity were identified in pairwise global alignments of the RSV L protein with its counterparts in four paramyxoviruses (parainfluenza virus type 3, Sendai virus, measles virus, Newcastle disease virus) and two rhabdoviruses (rabies virus, vesicular stomatitis virus). In addition, amino acid sequence alignments showed that the RSV L protein has a 70-amino acid amino-terminal extension relative to the others. This is suggested to be due to the acquisition of gene overlap of the RSV L gene with its upstream neighbor, the 22K (M2) gene and the use of a new translational start site. The most highly related region among these seven proteins is located within the amino-terminal half, representing approximately 20% of each protein sequences. This region contains six discrete segments that are colinear and highly conserved in each paramyxovirus and rhabdovirus L protein, and three of these overlapped with sequence motifs found previously in other RNA-dependent RNA and DNA polymerases. A phylogenetic tree was constructed from the paramyxovirus and rhabdovirus L protein sequences to further define their relationships. The branching order indicates that RSV represents a lineage within the paramyxovirus family which is relatively distinct from the others, which in turn are more closely interrelated. Among these other members of the family Paramyxoviridae, the branching order does not entirely conform to their current taxonomic organization, providing support for its reevaluation.

Antibody-selected variation and reversion in Sindbis virus neutralization epitopes.

Sindbis virus variants evidencing a complex and bidirectional tendency toward spontaneous antigenic change were isolated and characterized. Variants were selected on the basis of their escape from neutralization by individual monoclonal antibodies to either of the two envelope glycoproteins, E2 and E1. Multisite variants, including one altered in three neutralization sites, were obtained by selecting mutants consecutively in the presence of different neutralizing monoclonal antibodies. Two phenotypic revertants, each of which reacquired prototype antigenicity, were back-selected on the basis of their reactivity with a neutralizing monoclonal antibody. An incidental oligonucleotide marker distinguished these and the variant from which they arose from parental Sindbis virus and other mutants, thereby confirming that the revertants were true progeny of the antigenic variant. Prototype Sindbis virus and variants derived from it were compared on the basis of their reactivities with each of a panel of monoclonal antibodies; patterns revealed a minimum of five independently mutable Sindbis virus neutralization epitopes, segregating as three antigenic sites (two E2 and one E1).