Respiratory Syncytial Virus Genome Research Articles

Chimeric pneumovirus nucleocapsid (N) proteins allow identification of amino acids essential for the function of the respiratory syncytial virus N protein.

The nucleocapsid (N) protein of the pneumovirus respiratory syncytial virus (RSV) is a major structural protein which encapsidates the RNA genome and is essential for replication and transcription of the RSV genome. The N protein of the related virus pneumonia virus of mice (PVM) is functionally unable to replace the RSV N protein in a minigenome replication assay. Using chimeric proteins, in which the immediate C-terminal part of the RSV N protein was replaced with the equivalent region of the PVM N protein, it was shown that six amino acid residues near the C terminus of the N protein (between residues 352-369) are essential for its function in replication and for the ability of the N protein to bind to the viral phosphoprotein, P.

A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome: predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA

Human respiratory syncytial virus (HRSV) escape mutants selected with antibodies specific for the attachment (G) protein contain diverse genetic alterations, including point mutations, premature stop codons, frame shift changes and A to G hypermutations. The latter changes have only been found in mutants selected with antibodies directed against the conserved central region of the G protein. This gene segment fulfils substrate requirements for adenosine deaminases that act on RNA (ADARs): i.e. it is an A+U rich region of 137 residues, and 98 or 106 of them--for A/Mon/3/88 or Long HRSV strains, respectively--are predicted to form intramolecular base pairs leading to a stable RNA secondary structure. In addition, when sequences of the G gene from natural isolates are compared in terms of pairwise substitutions, A to G+G to A changes are preferentially observed in regions where stable intramolecular dsRNA secondary structures are predicted to occur. In this study, a model is proposed in which, in addition to nucleotide misincorporations, reiterative A to G changes in HRSV are generated by ADAR activity operating in short segments (100-200 ribonucleotide residues) of the HRSV genome with high tendency for intramolecular base pairing.

Targeted therapy of respiratory syncytial virus in African green monkeys by intranasally administered 2-5A antisense.

Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, young children, immunocompromised patients, and the institutionalized elderly. Previous work had shown that RNase L, an antiviral enzyme of the interferon system, could be recruited to cleave RSV genomic RNA by attaching tetrameric 2prime prime or minute-5prime prime or minute-linked oligoadenylates (2-5A) to an oligonucleotide complementary to repetitive gene-start sequences within the RSV genome (2-5A antisense). A 2prime prime or minute-O-methyl RNA-modified analog of the lead 2-5A anti-RSV chimera is shown here to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. When administered intranasally to RSV-infected African green monkeys, this chimera reduced nasal RSV replication by up to four log(10) units in a dose- and time-dependent manner.

RSV virus manipulates the human fractalkine receptor

Respiratory syncytial virus (RSV) is a common cause of upper respiratory tract infections, and in some children causes wheezing and potentially lethal damage to the lower respiratory tract. More-severe infections are accompanied by the release of largely T helper (Th)1-type inflammatory cytokines. It has proved difficult to engineer a vaccine to protect children at risk of serious illness as host–virus interactions are unclear. The RSV genome encodes 11proteins, of which only two induce antibodies capable of neutralizing virus–the F and Gglycoproteins.

Persistence of Respiratory Syncytial Virus in Macrophages Alters Phagocytosis and Pro-inflammatory Cytokine Production

Functions of macrophage are known to be altered by acute infection with respiratory syncytial virus (RSV). However, it is unknown whether the persistent presence and expression of the RSV genome have any effect on the functions of these cells. We used a murine macrophage-like cell line (P388D1) persistently infected with RSV to determine: (i) phagocytic activity mediated by Fcgamma receptors, (ii) expression of Fcgamma receptors, and (iii) production of IL-1beta, IL-6 and TNF-alpha. Viral persistence was found to increase phagocytosis, expression of Fcgamma receptors and the production of IL-1beta and IL-6. In contrast the biological activity of secreted TNF-alpha decreased. In this study we give novel evidence that RSV persistence alters the biological activities of macrophages.

Recombinant Respiratory Syncytial Viruses with Deletions in the NS1, NS2, SH, and M2-2 Genes Are Attenuated in Vitro and in Vivo

Respiratory syncytial virus (RSV) encodes several proteins that lack well-defined functions; these include NS1, NS2, SH, and M2-2. Previous work has demonstrated that NS2, SH, and M2-2 can each be deleted from RSV genome and thus are considered as accessory proteins. To determine whether RSV can replicate efficiently when two or more transcriptional units are deleted, we removed NS1, NS2, SH, and M2-2 genes individually and in different combinations from an infectious cDNA clone derived from human RSV A2 strain. The following six mutants with two or more genes deleted were obtained: ΔNS1NS2, ΔM2-2SH, ΔM2-2NS2, ΔSHNS1, ΔSHNS2, and ΔSHNS1NS2. Deletion of M2-2 together with NS1 was detrimental to RSV replication. It was not possible to obtain a recombinant RSV when all four genes were deleted. All of the double and triple deletion mutants exhibited reduced replication and small plaque morphology in vitro. Replication of these deletion mutants was more reduced in HEp-2 cells than in Vero cells. Among the 10 single and multiple gene deletion mutants obtained, ΔM2-2NS2 was most attenuated. ΔM2-2NS2 formed barely visible plaques in HEp-2 cells and had a reduction of titer of 3 log10 compared with the wild-type recombinant RSV in infected HEp-2 cells. When inoculated intranasally into cotton rats, all of the deletion mutants were attenuated in the respiratory tract. Our data indicated that the NS1, NS2, SH, and M2-2 proteins, although dispensable for virus replication in vitro, provide auxiliary functions for efficient RSV replication.

Profilin is required for optimal actin-dependent transcription of respiratory syncytial virus genome RNA.

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-L-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-L-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-L-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.

RECOVERY OF RESPIRATORY SYNCYTIAL VIRUS FROM STETHOSCOPES BY CONVENTIONAL VIRAL CULTURE AND POLYMERASE CHAIN REACTION

RECOVERY OF RESPIRATORY SYNCYTIAL VIRUS FROM STETHOSCOPES BY CONVENTIONAL VIRAL CULTURE AND POLYMERASE CHAIN REACTION

Recombinant Human Respiratory Syncytial Virus (RSV) from cDNA and Construction of Subgroup A and B Chimeric RSV

Infectious human respiratory syncytial virus (RSV) was produced from a cDNA clone that contains 15,222 nucleotides of RSV genome derived from the A2 strain of subgroup A. Recovery of infectious RSV from cDNA required cotransfection of only three expression plasmids encoding the nucleoprotein (N), the phosphoprotein (P), and the major polymerase protein (L). Inclusion of the M2–1 plasmid was not required in the transfection reaction and if included did not significantly increase the rescue efficiency. However, a single nucleotide substitution in the RSV leader region (C to G at position 4 in the antigenomic sense), greatly increased the amount of infectious virus recovered from cDNA. A recombinant RSVA2 virus that expresses an additional structural G protein derived from a subgroup B RSV was also obtained. Both A2 and B strain G glycoproteins were expressed in cells infected with the chimeric RSV. A chimeric RSV that expresses a heterologous subgroup antigen in a live attenuated vaccine candidate may be important for prevention of diseases associated with both RSV subgroup A and subgroup B infection.

Analysis of respiratory syncytial virus in clinical samples by reverse transcriptase-polymerase chain reaction restriction mapping.

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

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(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms 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.

Optimization of PCR and automated sequencing of clinical isolates of respiratory syncytial virus

A streamlined protocol was developed to carry out nucleotide sequence analysis on PCR products obtained from regions of the RNA genome of respiratory syncytial virus (RSV). Whole cell RNA extracted from tissue culture cells inoculated with RSV-infected nasopharyngeal aspirates was used as a template for single-tube cDNA synthesis and PCR amplification of specific regions of the RSV fusion (F) protein gene. The products were then purified using Sephacryl spin columns and digested using λ-exonuclease to obtain single-stranded templates for analysis using dye-primer chemistry in an automated sequencer. Optimization of a range of parameters and comparison of a number of alternate procedures resulted in a final protocol that allowed the comparison of the sequences of a panel of local RSV subgroup A and B clinical isolates.

Persistence of Respiratory Syncytial Virus Genome and Protein After Acute Bronchiolitis in Guinea Pigs

Children with acute respiratory syncytial virus (RSV) bronchiolitis often develop sequelae of recurrent wheezing and asthma. To determine whether RSV persists within the lung after resolution of acute bronchiolitis, we examined the lungs of guinea pigs 60 days after intranasal inoculation with either human RSV (n = 10) or uninfected cell culture supernatant (n = 11). Evidence of viral persistence within the lung was determined by viral culture to test for replicating virus, immunohistochemistry to test for viral protein, and the reverse transcriptase-polymerase chain reaction (RT-PCR) to test for viral genomic RNA. Lungs were also examined histologically for evidence of bronchiolar inflammation or increased numbers of mast cells in the airway walls. All viral cultures were negative; however, there was positive immunohistochemical staining of occasional alveolar macrophages in six of ten RSV-inoculated guinea pigs while RT-PCR was positive in seven of ten RSV-inoculated animals. The six guinea pigs with evidence of RSV by immunohistochemistry and RT-PCR showed excess bronchiolar polymorphonuclear cell infiltrates (p < 0.005) but no increase in the number of airway wall mast cells. These results show that RSV protein and genomic RNA can persist in the lungs of experimentally inoculated guinea pigs for at least 60 days after infection and that persistence of the virus within alveolar macrophages might contribute to the pathogenesis of chronic bronchiolar inflammation.

Nucleotide and predicted amino acid sequence analysis of the ovine respiratory syncytial virus non-structural 1C and 1B genes and the small hydrophobic protein gene.

Respiratory syncytial virus (RSV) infects humans and cattle causing serious respiratory tract disease in both. The genome of the human and bovine RSV (HRSV and BRSV) codes for two non-structural and eight structural proteins. RSV has also been isolated from naturally infected sheep, but the genome of the ovine RSV (ORSV) has not been characterized and nor has the virus host range been identified. Here we report on the cloning and sequencing of the two non-structural 1C and 1B genes and of the small hydrophobic (SH) protein gene of the ORSV. The nucleotide identity of the ORSV 1C gene to those of subgroups A and B of HRSV was 70% and 65% respectively whereas the predicted amino acid identity was 68% and 69% respectively. The ORSV 1B gene had a 70% and 72% nucleotide identity with that of subgroups A and B of the HRSV respectively, and 79% predicted amino acid identity with both HRSV subgroups. The identity level of these two ORSV 1C and 1B genes to those of the BRSV could not be determined since these two BRSV genes have not been sequenced. The SH protein of RSV is a structural protein expressed on the surface of infected cells. In common with HRSV and BRSV subgroups, the ORSV SH had the three proposed domains with the C-terminal domain least conserved among the viruses. In the latter domain, the ORSV SH gene had a nucleotide identity of 68 to 69% and 47 to 51% with those of the BRSV and HRSV, respectively, and a predicted amino acid identity of 56% and 33 to 47% with those of BRSV and HRSV, respectively. Defining the genes and their products should help determine which genes and gene products are most suitable for use as diagnostic tools or as vaccine candidates.

Presence of Respiratory Syncytial Virus Genomic Sequences in Middle Ear Fluid and Its Relationship to Expression of Cytokines and Cell Adhesion Molecules

The presence of respiratory syncytial virus (RSV) and several cytokines and cell adhesion molecules in middle ear effusions and mucosal tissues was evaluated using polymerase chain reaction. RSV genomic sequences were detected in 23 (52.7%) of 44 middle ear effusions tested. The sequences were detectable at an even higher rate (82.4%) in effusions of children in whom infectious virus was detected in the nasopharynx. All samples with the RSV genome contained the mRNA for interleukin-1 beta and -6 and tumor necrosis factor-alpha. The messages for these cytokines, together with intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1, were detected in human middle ear mucosal organ cultures infected in vitro with RSV. Our results suggest that the enhanced synthesis of proinflammatory cytokines and cell adhesion molecules in the middle ear infected with RSV may contribute to the inflammatory processes in otitis media.

Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s).

Extracts made from human respiratory syncytial virus (RSV)-infected Hep-2 cells synthesized mRNAs encoded by all known viral genes. In contrast, RSV ribonucleoproteins purified from infected cells failed to transcribe in vitro; transcription was restored by addition of a cytoplasmic extract of uninfected Hep-2 cells, demonstrating that a cellular factor(s) has a role in RSV gene expression. Quantitation of the individual gene mRNAs transcribed in vitro revealed polarity of transcription of the genome.

Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification.

A rapid method for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates, involving a combination of reverse transcription and polymerase chain reaction amplification (RT-PCR), has been developed. The RT-PCR assay employs oligonucleotide primers specific for the region of the RSV genome which encodes the F1 subunit of the fusion (F) glycoprotein. Other respiratory viruses do not give a positive reaction. The RT-PCR assay was tested on 202 nasopharyngeal aspirates collected from children with clinical signs of respiratory infection, and the results from RT-PCR were compared with those obtained from virus culture and direct detection by enzyme immunoassay (EIA). RT-PCR results were positive in 118 of 125 samples from which RSV was cultured, as well as in 4 of 7 samples which were culture negative but EIA positive. RT-PCR results were negative in 68 of 70 culture-negative, EIA-negative samples, which included 11 samples from which other respiratory viruses were isolated. The speed, sensitivity (94.6%), and specificity (greater than 97%) of the RT-PCR assay suggest that this technique could be useful for rapid detection of RSV in clinical samples.

Structural aspect of the respiratory syncytial virus genome

Structural aspect of the respiratory syncytial virus genome

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.