Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation

Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate.

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Local Evolutionary Patterns of Human Respiratory Syncytial Virus Derived from Whole-Genome Sequencing

Journal of Virology ◽

10.1128/jvi.03391-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3444-3454 ◽

Cited By ~ 41

Author(s):

Charles N. Agoti ◽

James R. Otieno ◽

Patrick K. Munywoki ◽

Alexander G. Mwihuri ◽

Patricia A. Cane ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Large Scale ◽

Vaccine Development ◽

Human Respiratory Syncytial Virus ◽

Genomic Variation ◽

Genomic Sequences ◽

The Novel ◽

Sequencing Method ◽

The World ◽

Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies.IMPORTANCEThe new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

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Genetic Variability and Molecular Evolution of the Human Respiratory Syncytial Virus Subgroup B Attachment G Protein

Journal of Virology ◽

10.1128/jvi.79.14.9157-9167.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 9157-9167 ◽

Cited By ~ 97

Author(s):

Kalina T. Zlateva ◽

Philippe Lemey ◽

Elien Moës ◽

Anne-Mieke Vandamme ◽

Marc Van Ranst

Keyword(s):

Respiratory Syncytial Virus ◽

Genetic Variability ◽

G Protein ◽

Antigenic Variation ◽

Human Respiratory Syncytial Virus ◽

Recent Common Ancestor ◽

Nucleotide Substitutions ◽

Host Immune Responses ◽

Most Recent Common Ancestor ◽

Syncytial Virus

ABSTRACT Human respiratory syncytial virus (HRSV) is the most important cause of acute respiratory disease in infants. Two major subgroups (A and B) have been identified based on antigenic differences in the attachment G protein. Antigenic variation between and within the subgroups may contribute to reinfections with these viruses by evading the host immune responses. To investigate the circulation patterns and mechanisms by which HRSV-B viruses evolve, we analyzed the G protein genetic variability of subgroup B sequences isolated over a 45-year period, including 196 Belgian strains obtained over 22 epidemic seasons (1982 to 2004). Our study revealed that the HRSV-B evolutionary rate (1.95 × 10−3 nucleotide substitutions/site/year) is similar to that previously estimated for HRSV-A (1.83 × 10−3 nucleotide substitutions/site/year). However, natural HRSV-B isolates appear to accommodate more drastic changes in their attachment G proteins. The most recent common ancestor of the currently circulating subgroup B strains was estimated to date back to around the year 1949. The divergence between the two major subgroups was calculated to have occurred approximately 350 years ago. Furthermore, we have identified 12 positively selected sites in the G protein ectodomain, suggesting that immune-driven selective pressure operates in certain codon positions. HRSV-A and -B strains have similar phylodynamic patterns: both subgroups are characterized by global spatiotemporal strain dynamics, where the high infectiousness of HRSV permits the rapid geographic spread of novel strain variants.

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Evolution of Human Respiratory Syncytial Virus (RSV) over Multiple Seasons in New South Wales, Australia

10.20944/preprints201808.0318.v1 ◽

2018 ◽

Author(s):

Francesca Di Giallonardo ◽

Jen Kok ◽

Marian Fernandez ◽

Ian Carter ◽

Jemma L. Geoghegan ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

New South ◽

New South Wales ◽

Host Population ◽

Human Respiratory Syncytial Virus ◽

Nucleotide Substitutions ◽

South Wales ◽

A Genome ◽

Syncytial Virus ◽

Tract Infections

There is an ongoing global pandemic of human respiratory syncytial virus (RSV) infection that results in substantial annual morbidity and mortality. In Australia, RSV is the major cause of acute lower respiratory tract infections (ALRI). Nevertheless, little is known about the extent and origins of genetic diversity of RSV in Australia, nor the factors that shape this diversity. We conducted a genome-scale analysis of RSV infections in New South Wales (NSW). RSV genomes were successfully sequenced for 144 specimens collected between 2010-2016. Of these, 64 belonged to the RSVA and 80 to the RSVB subtype. Phylogenetic analysis revealed a wide diversity of RSV lineages within NSW and that both subtypes evolved rapidly in a strongly clock-like manner, with mean rates of approximately 6-8 x 10-4 nucleotide substitutions per site per year. There was only weak evidence for geographic clustering of sequences, indicative of fluid patterns of transmission within the infected population, and no evidence of any clustering by patient age such that viruses in the same lineages circulate through the entire host population. Importantly, we show that both subtypes circulated concurrently in NSW with multiple introductions into the Australian population in each year, and only limited evidence for multi-year persistence.

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Molecular Evolution and Circulation Patterns of Human Respiratory Syncytial Virus Subgroup A: Positively Selected Sites in the Attachment G Glycoprotein

Journal of Virology ◽

10.1128/jvi.78.9.4675-4683.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4675-4683 ◽

Cited By ~ 82

Author(s):

Kalina T. Zlateva ◽

Philippe Lemey ◽

Anne-Mieke Vandamme ◽

Marc Van Ranst

Keyword(s):

Respiratory Syncytial Virus ◽

Hypervariable Region ◽

Human Respiratory Syncytial Virus ◽

Likelihood Method ◽

Recent Common Ancestor ◽

Nucleotide Substitutions ◽

Most Recent Common Ancestor ◽

Syncytial Virus ◽

Positively Selected Sites

ABSTRACT Human respiratory syncytial virus (HRSV) is the most common etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. In this study, we have analyzed nucleotide sequences encompassing 629 bp at the carboxy terminus of the G glycoprotein gene for HRSV subgroup A strains isolated over 47 years, including 112 Belgian strains isolated over 19 consecutive years (1984 to 2002). By using a maximum likelihood method, we have tested the presence of diversifying selection and identified 13 positively selected sites with a posterior probability above 0.5. The sites under positive selection correspond to sites of O glycosylation or to amino acids that were previously described as monoclonal antibody-induced in vitro escape mutants. Our findings suggest that the evolution of subgroup A HRSV G glycoprotein is driven by immune pressure operating in certain codon positions located mainly in the second hypervariable region of the ectodomain. Phylogenetic analysis revealed the prolonged cocirculation of two subgroup A lineages among the Belgian population and the possible extinction of three other lineages. The evolutionary rate of HRSV subgroup A isolates was estimated to be 1.83 × 10−3 nucleotide substitutions/site/year, projecting the most recent common ancestor back to the early 1940s.

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Genetic Stability of Parainfluenza Virus 5-Vectored Human Respiratory Syncytial Virus Vaccine Candidates after In Vitro and In Vivo Passage

Journal of Virology ◽

10.1128/jvi.00559-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 6

Author(s):

Shannon I. Phan ◽

Carolyn M. Adam ◽

Zhenhai Chen ◽

Michael Citron ◽

Xiaoping Liang ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Genetic Stability ◽

Human Respiratory Syncytial Virus ◽

Parainfluenza Virus ◽

Vaccine Candidates ◽

Consensus Sequences ◽

Parainfluenza Virus 5 ◽

Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome. However, the function of the foreign gene insertion may need to be considered when designing PIV5-based vaccines. IMPORTANCE The genetic stability of live viral vaccines is important for safety and efficacy. PIV5 is a promising live viral vector and has been used to develop vaccines. In this work, we examined the genetic stability of a PIV5-based RSV vaccine in vitro and in vivo. We found that insertions of foreign genes, such as the RSV F and G genes, were stably maintained in the PIV5 genome and there was no mutation that abolished the expression of RSV F or G. Interestingly, the function of the inserted gene may have an impact on PIV5 genome stability.

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Large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in Beijing, China

Virology Journal ◽

10.1186/1743-422x-8-62 ◽

2011 ◽

Vol 8 (1) ◽

pp. 62 ◽

Cited By ~ 24

Author(s):

Guilan Lu ◽

Richard Gonzalez ◽

Li Guo ◽

Chao Wu ◽

Jiang Wu ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Large Scale ◽

Human Metapneumovirus ◽

Human Respiratory Syncytial Virus ◽

Virus Infections ◽

Respiratory Syncytial Virus Infections ◽

Syncytial Virus ◽

Beijing China

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Evolution of Human Respiratory Syncytial Virus (RSV) over Multiple Seasons in New South Wales, Australia

Viruses ◽

10.3390/v10090476 ◽

2018 ◽

Vol 10 (9) ◽

pp. 476 ◽

Cited By ~ 10

Author(s):

Francesca Di Giallonardo ◽

Jen Kok ◽

Marian Fernandez ◽

Ian Carter ◽

Jemma Geoghegan ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

New South ◽

New South Wales ◽

Host Population ◽

Human Respiratory Syncytial Virus ◽

Nucleotide Substitutions ◽

South Wales ◽

A Genome ◽

Syncytial Virus ◽

Tract Infections

There is an ongoing global pandemic of human respiratory syncytial virus (RSV) infection that results in substantial annual morbidity and mortality. In Australia, RSV is a major cause of acute lower respiratory tract infections (ALRI). Nevertheless, little is known about the extent and origins of the genetic diversity of RSV in Australia, nor the factors that shape this diversity. We have conducted a genome-scale analysis of RSV infections in New South Wales (NSW). RSV genomes were successfully sequenced for 144 specimens collected between 2010–2016. Of these, 64 belonged to the RSVA and 80 to the RSVB subtype. Phylogenetic analysis revealed a wide diversity of RSV lineages within NSW and that both subtypes evolved rapidly in a strongly clock-like manner, with mean rates of approximately 6–8 × 10−4 nucleotide substitutions per site per year. There was only weak evidence for geographic clustering of sequences, indicative of fluid patterns of transmission within the infected population and no evidence of any clustering by patient age such that viruses in the same lineages circulate through the entire host population. Importantly, we show that both subtypes circulated concurrently in NSW with multiple introductions into the Australian population in each year and only limited evidence for multi-year persistence.

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