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.