RESUMO
Respiratory syncytial virus is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroup, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the US. In-depth analysis and annotation of the protein variants in L and the other replication-associated proteins N, P, M2-1, and M2-2 identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its co-factor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates.
RESUMO
RNA viruses generate nonstandard viral genomes during their replication, including viral genomes of the copy-back (cbVGs) type that cannot replicate in the absence of a standard virus. cbVGs play a crucial role in shaping virus infection outcomes due to their ability to interfere with virus replication and induce strong immune responses. However, despite their critical role during infection, the principles that drive the selection and evolution of cbVGs within a virus population are poorly understood. As cbVGs are dependent on the virus replication machinery to be generated and replicated, we hypothesized that host factors that affect virus replication exert selective pressure on cbVGs and drive their evolution within a virus population. To test this hypothesis, we used respiratory syncytial virus (RSV) as a model and took an experimental evolution approach by serially passaging RSV in immune-competent human lung adenocarcinoma A549 control and immune-deficient A549 Signal transducer and activator of transcription 1 (STAT1) KO cells, which allow higher levels of virus replication. As predicted, we observed that virus populations accumulated higher amounts of cbVGs in the more permissive A549 STAT1 KO cells over time; however, unexpectedly, the predominant cbVG species after passages in the two conditions were different. While A549 STAT1 KO cells accumulated relatively short cbVGs, A549 control cells mainly contained cbVGs of much longer predicted size, which have not been described previously. These long cbVGs were predominant at first in both cell lines in vitro and the predominant ones observed in samples from RSV-infected patients. Although sustained high replication levels are associated with cbVG generation and accumulation, our data show that sustained high levels of virus replication are critical for cbVG population diversification, a process that precedes the generation of shorter cbVGs that selectively accumulate over time. Taken together, we show that selection and evolution of cbVGs within a virus population are shaped by how resistant or permissive a host is to RSV.
