A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein.

UNLABELLED: Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. IMPORTANCE: RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats.

UNLABELLED: Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. IMPORTANCE: RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

Respiratory syncytial virus infection activates IL-13-producing group 2 innate lymphoid cells through thymic stromal lymphopoietin.

BACKGROUND: Respiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated illness is in part caused by immunopathology associated with a robust type 2 response. OBJECTIVE: We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. METHODS: Wild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential. RESULTS: RSV induced a 3-fold increase in the number of IL-13-producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13-producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13-producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13-producing ILC2s through a TSLP-dependent mechanism. CONCLUSION: These data demonstrate that multiple pathogenic strains of RSV induce IL-13-producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection.

Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein.

UNLABELLED: There are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup, isolates are further divided into clades. Several years ago, multiple subgroup B isolates which contained a duplication of 60 nucleotides in the glycoprotein (G) gene were described. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene or a consensus BA G gene lacking the duplication (GΔdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness. IMPORTANCE: Respiratory syncytial virus (RSV) is an important pathogen for infants for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation.

Identification of residues in the human respiratory syncytial virus fusion protein that modulate fusion activity and pathogenesis.

UNLABELLED: Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein. IMPORTANCE: Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.

The respiratory syncytial virus fusion protein targets to the perimeter of inclusion bodies and facilitates filament formation by a cytoplasmic tail-dependent mechanism.

The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.

Optimization of Bangladesh and Malaysian genotype recombinant reporter Nipah viruses for in vitro antiviral screening and in vivo disease modeling.

Nipah virus (NiV) causes near-annual outbreaks of fatal encephalitis and respiratory disease in South Asia with a high mortality rate (∼70%). Since there are no approved therapeutics for NiV disease in humans, the WHO has designated NiV and henipaviral diseases priority pathogens for research and development. We generated a new recombinant green fluorescent reporter NiV of the circulating Bangladesh genotype (rNiV-B-ZsG) and optimized it alongside our previously generated Malaysian genotype reporter counterpart (rNiV-M-ZsG) for antiviral screening in primary-like human respiratory cell types. Validating our platform for rNiV-B-ZsG with a synthetic compound library directed against viral RNA-dependent RNA polymerases, we identified a hit compound and confirmed its sub-micromolar activity against wild-type NiV, green fluorescent reporter, and the newly constructed bioluminescent red fluorescent double reporter (rNiV-B-BREP) NiV. We furthermore demonstrated that rNiV-B-ZsG and rNiV-B-BREP viruses showed pathogenicity comparable to wild-type NiV-B in the Syrian golden hamster model of disease, supporting additional use of these tools for both pathogenesis and advanced pre-clinical studies in vivo.

A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function.

Nipah virus is a highly lethal zoonotic pathogen found in Southeast Asia that has caused human encephalitis outbreaks with 40%-70% mortality. NiV encodes its own RNA-dependent RNA polymerase within the large protein, L. Efficient polymerase activity requires the phosphoprotein, P, which tethers L to its template, the viral nucleocapsid. P is a multifunctional protein with modular domains. The central P multimerization domain is composed of a long, tetrameric coiled coil. We investigated the importance of structural features found in this domain for polymerase function using a newly constructed NiV bicistronic minigenome assay. We identified a conserved basic patch and central kink in the coiled coil that are important for polymerase function, with R555 being absolutely essential. This basic patch and central kink are conserved in the related human pathogens measles and mumps viruses, suggesting that this mechanism may be conserved.

BAC-Based Recovery of Recombinant Respiratory Syncytial Virus (RSV).

Respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus, which causes lower respiratory tract infections and is a leading cause of mortality in young infants. There is no available RSV vaccine and currently administered prophylactic antibodies are limited to high-risk populations. Current efforts to develop vaccines include development of live-attenuated RSV candidates. We describe here methods for preparation and recovery of recombinant RSV using an efficient bacterial artificial chromosome (BAC)-based system, expansion and plaque purification of recovered virus, and generation of master and working stocks.

4'-Azidocytidine (R1479) inhibits henipaviruses and other paramyxoviruses with high potency.

The henipaviruses Nipah virus and Hendra virus are highly pathogenic zoonotic paramyxoviruses which have caused fatal outbreaks of encephalitis and respiratory disease in humans. Despite the availability of a licensed equine Hendra virus vaccine and a neutralizing monoclonal antibody shown to be efficacious against henipavirus infections in non-human primates, there remains no approved therapeutics or vaccines for human use. To explore the possibility of developing small-molecule nucleoside inhibitors against henipaviruses, we evaluated the antiviral activity of 4'-azidocytidine (R1479), a drug previously identified to inhibit flaviviruses, against henipaviruses along with other representative members of the family Paramyxoviridae. We observed similar levels of R1479 antiviral activity across the family, regardless of virus genus. Our brief study expands the documented range of viruses susceptible to R1479, and provides the basis for future investigation and development of 4'-modified nucleoside analogs as potential broad-spectrum antiviral therapeutics across both positive and negative-sense RNA virus families.

GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Structural basis of respiratory syncytial virus subtype-dependent neutralization by an antibody targeting the fusion glycoprotein.

A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines.

Reverse Genetics of Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a negative-strand RNA virus that is associated with severe lower respiratory tract infections in young infants and the elderly. RSV remains a leading cause worldwide of infant mortality, and despite the high clinical and economic burden of the virus there are currently no available vaccines. Here, we describe the methods for recovery of recombinant RSV viruses using a bacterial artificial chromosome and methods related to procurement and expansion of stocks of RSV mutants.

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.

Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes.

UNLABELLED: Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in children for which there is no licensed vaccine. Live-attenuated RSV vaccines are the most clinically advanced in children, but achieving an optimal balance of attenuation and immunogenicity is challenging. One way to potentially retain or enhance immunogenicity of attenuated virus is to mutate virulence genes that suppress host immune responses. The NS1 and NS2 virulence genes of the RSV A2 strain were codon deoptimized according to either human or virus codon usage bias, and the resulting recombinant viruses (dNSh and dNSv, respectively) were rescued by reverse genetics. RSV dNSh exhibited the desired phenotype of reduced NS1 and NS2 expression. RSV dNSh was attenuated in BEAS-2B and primary differentiated airway epithelial cells but not in HEp-2 or Vero cells. In BALB/c mice, RSV dNSh exhibited a lower viral load than did A2, and yet it induced slightly higher levels of RSV-neutralizing antibodies than did A2. RSV A2 and RSV dNSh induced equivalent protection against challenge strains A/1997/12-35 and A2-line19F. RSV dNSh caused less STAT2 degradation and less NF-κB activation than did A2 in vitro. Serial passage of RSV dNSh in BEAS-2B cells did not result in mutations in the deoptimized sequences. Taken together, RSV dNSh was moderately attenuated, more immunogenic, and equally protective compared to wild-type RSV and genetically stable. IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of infant viral death in the United States and worldwide, and no vaccine is available. Live-attenuated RSV vaccines are the most studied in children but have suffered from genetic instability and low immunogenicity. In order to address both obstacles, we selectively changed the codon usage of the RSV nonstructural (NS) virulence genes NS1 and NS2 to the least-used codons in the human genome (deoptimization). Compared to parental RSV, the codon-deoptimized NS1/NS2 RSV was attenuated in vitro and in mice but induced higher levels of neutralizing antibodies and equivalent protection against challenge. We identified a new attenuating module that retains immunogenicity and is genetically stable, achieved through specific targeting of nonessential virulence genes by codon usage deoptimization.

A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis.

We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered two RSV reporter viruses, one expressing the red fluorescent protein monomeric Katushka 2 (A2-K-line19F) and one expressing Renilla luciferase (A2-RL-line19F). As proof of principle, we efficiently generated a RSV gene deletion mutant (A2-line19FΔNS1/NS2) and a point mutant (A2-K-line19F-I557V) by recombination-mediated BAC mutagenesis. Together with sequence-optimized helper expression plasmids, BAC-RSV is a stable, versatile, and efficient reverse genetics platform for generation of a recombinant Pneumovirus.