Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(0RF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(0RF1) protein is consistent with its recent identification as a transcription elongation factor and contirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.Human respiratory syncytial virus (RSV) is the most important pediatric viral respiratory pathogen worldwide (1-3). This ubiquitous highly infectious agent emerges each year in seasonal epidemics and nearly everyone is infected at least once within the first 2 years of life. RSV disease is responsible for considerable morbidity and mortality and lacks an approved vaccine or highly effective antiviral therapy. Research on RSV is impeded by its poor growth in tissue culture, the instability of the virion, and the lack of a highly permissive experimental animal other than the chimpanzee.Resistance to RSV reinfection induced by natural infection is incomplete but increases incrementally with repeated exposure. Thus, RSV can infect multiple times during childhood and later life, but serious disease usually is limited to the first and sometimes second infections of life. The minimum goal of RSV immunoprophylaxis is to induce sufficient resistance to prevent serious disease associated with the first or second infection.RSV is a member of the pneumovirus genus of the paramyxovirus family (1, 4 The development of methods for introducing designed changes into genomic RNA of nonsegmented negative-strand RNA viruses was impeded by the lack of homologous viral recombination and the lack of infectivity of naked genomic RNA. The supposition that the minimum unit of infectivity for this type of virus is a nucleocapsid competent for RNA synthesis suggested a different strategy to produce infectious virus from viral cDNA. This involved the intracellular coexpression, from separate transfected plasmids, of cDNAencoded genomic or antigenomic RNA and those viral proteins necessary to generate a transcribing and replicating nucleocapsid. cDNA expression would be driven by T7 RNA polymerase supplied by a vaccinia recombinant virus. This approach was developed first by using short internally deleted analogs of genomic or antigenomic RNA ("minigenomes") that were shown to participate in...

Active immunity and maternally transmitted passive immunity to respiratory syncytial virus (RSV) were studied in cotton rats. Animals infected with respiratory syncytial virus developed complete resistance to pulmonary reinfection, which lasted at least 18 months. Nasal resistance was of shorter duration and began to diminish in 8 months. Pulmonary resistance was transferred by parabiosis, but nasal resistance was not. Adoptive transfer studies with fractionated convalescent blood showed that serum antibody, but not circulating lymphocytes, conferred pulmonary resistance. Immune females conferred antibody to their young prenatally and postnatally, with most of the antibody being transferred via colostrum and milk. Maternally transmitted immunity was more effective in the lungs than in the nose and was transient in both organs. Foster nursing experiments showed colostrum and milk to be the most important routes of immune transfer. Although resistance in infants generally correlated with serum neutralizing antibody levels, several exceptions to this correlation suggested that immune factors other than neutralizing antibody may also play an important role in maternal passive immunity.

Complete sequences for the intergenic regions of the genome of human respiratory syncytial virus were obtained by dideoxynucleotide sequencing using synthetic oligonucleotides. These experiments established that the 10 respiratory syncytial viral genes are arranged, without additional intervening genes, in the order 3' IC-IB-N-P-M-IA-G-F-22K-L 5'. For the fi-rst nine genes, the exact gene boundaries were identified by comparison of the genomic sequences with previously determined mRNA sequences. The intergenic regions varied in length from 1 to 52 nucleotides and lacked any obvious conserved features of primary or secondary structure except that each sequence ended (3' to 5') with an adenosine residue. The exact start site of the 10th gene, the L gene, was not determined. However, RNA blot hybridization using a synthetic oligonucleotide designed from the genomic sequence mapped the L gene to within 54 nucleotides of the end of the penultimate 22K gene. The lack of conservation of chain length and nucleotide sequence for the respiratory syncytial viral intergenic regions, together with the complexity of the genetic map, contrasts with previous observations for other nonsegmented negative-strand viruses.Human respiratory syncytial (RS) virus, a pneumovirus of the paramyxovirus family, is a pleomorphic, enveloped, RNA-containing virus that is a primary cause of severe pediatric respiratory tract disease (1). Efforts to characterize the viral genome and gene products have been complicated by the instability of the virus and its poor growth in tissue culture.Ten viral mRNAs were identified by cDNA cloning and RNA blot hybridization analysis of infected-cell mRNAs (2, 3). The polypeptide coding assignments of the 10 mRNAs were identified by their individual translation in vitro (3). Complete cDNAs and nucleotide sequences have been obtained for 9 of the 10 viral mRNAs, excluding only the L mRNA (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Each mRNA appears to encode a single major polypeptide. The 10 RS viral proteins identified to date are the major nucleocapsid protein (N, 42.6 The RS viral genome is a single negative-strand of RNA of approximately 15,000 nucleotides (14), approximately equal to the combined sizes of the 10 viral mRNAs. The viral genome appears to be transcribed sequentially from a single promoter site, based on the kinetics of UV-inactivation of gene transcription (15). A viral transcriptional map, 3' IC-JB-N-P-M-1A-G-F-22K-L 5', was deduced from the UV mapping studies and from analysis of the relationships between the 10 mRNAs and a number of polycistronic readthrough mRNAs identified as minor products of viral transcription (2,3). However, these experimental approaches were inexact and provided no direct or detailed information on the structure of the viral genome.In the work described here, the intergenic and flanking gene sequences in genomic RNA were determined by the dideoxynucleotide method. This approach was designed to (i) confirm the sequences of the gene termini, deduced previously from ...

Four species of nonhuman primates were inoculated intranasally with 10(3.1) to 10(3.7) plaque forming units (pfu) of respiratory syncytial (RS) virus. Adults squirrel monkeys and newborn rhesus monkeys became infected and shed small quantities (peak titer 10(2.0) pfu/ml of nasopharyngeal swab specimen) of virus, but illness did not develop. Infant cebus monkeys aged 2 months became infected, shed 10(2.3) to 10(3.8) pfu/ml of nasopharyngeal swab specimen, but did not become ill. Chimpanzees aged 15 to 18 months shed a large quantity of virus, up to 10(6.0) pfu/ml of nasopharyngeal swab specimen and developed an upper respiratory illness. Chimpanzees are proposed as a possible animal model for future study of the immunopathology of RS virus disease and for in vivo evaluation of attenuated live virus vaccine candidates.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.

No abstract

An enzyme-linked immunosorbent assay was applied to the detection of serum antibodies against respiratory syncytial virus. The end points of the various sera tested in the assay were approximately 100 times higher than in the complementfixation test and 2 to 4 times higher than in the plaque reduction test. In addition, the immunosorbent assay appeared to be more efficient than the plaque reduction and complement-fixation techniques for detecting a serological response in young infants (1 to 6 months old) with serious respiratory syncytial virus lower respiratory disease. The simplicity, sensitivity, and rapidity of the enzyme-linked immunosorbent assay make it a useful tool for immunological studies with respiratory syncytial virus.

A previous report described the recovery from cDNA of infectious recombinant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Proc. Natl. Acad. Sci. USA, 92:11563-11567, 1995). Here, the system was used to construct recombinant RSV containing an additional gene encoding chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs, the transcription signals for the viral RNAdependent RNA polymerase. The RSV-CAT chimeric transcription cassette was inserted into the region between the G and F genes of the complete cDNA-encoded positive-sense RSV antigenome, and infectious CAT-expressing recombinant RSV was recovered. Transcription of the inserted gene into the predicted subgenomic polyadenylated mRNA was demonstrated by Northern (RNA) blot hybridization analysis, and the encoded protein was detected by enzyme assay and by radioimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild-type recombinant RSV. Consistent with this finding, the CAT-containing and wild-type viruses were very similar with regard to the levels of synthesis of the major viral proteins. Each of 25 RSV isolates obtained by plaque purification following eight serial passages expressed CAT, showing that the foreign gene was faithfully maintained in functional form. Analysis by reverse transcription and PCR did not reveal evidence of deletion of the foreign sequence. This finding demonstrated that the RSV genome can accept and maintain an increase in length of 762 nucleotides of foreign sequence and can be engineered to encode an additional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in virus growth and 20-fold decrease in virus yield in vitro. Thus, introduction of an additional gene into the RSV genome might represent a method of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.