Viruses are diverse pathogens that use host factors to enter cells and cause disease. Imaging the entry and replication phases of viruses and their interactions with host factors is key to fully understanding viral infections. This review will discuss how confocal microscopy and imaging flow cytometry are used to investigate virus entry and replication mechanisms in fixed and live cells. Quantification of viral images and the use of cryo-electron microscopy to gather structural information of viruses is also explored. Using imaging to understand how viruses replicate and interact with host factors, we gain insight into cellular processes and identify novel targets to develop antiviral therapeutics and vaccines.
Respiratory syncytial virus (RSV) infection is the most common cause of infant hospitalizations worldwide, and in the absence of an RSV vaccine, antibody prophylaxis is the only means of protection. The two types of RSV, RSVA and RSVB, share a high level of amino acid identity but they have different serum neutralization profiles. Here, we describe a single amino acid at position 305 in the RSV fusion glycoprotein (RSV-F) that switches the profile from RSVA (Leucine-L) to RSVB (Isoleucine-I). We found that the L305I mutation caused protein-wide shape-shift, altering both antibody and patient serum neutralization susceptibility. We show that viral replication in the presence of selective pressure forces mutation at position 305 and was the only amino acid mutation to occur in RSVA at evolutionary bottlenecks. We also identified a new mutation in RSVB that was associated with pressure from antibody neutralization. We engineered the L305I mutation in an RSV reverse genetic model system that led to a switch in susceptibility between RSVA and B patient sera. We postulate that this event was a significant determinant in the divergence of RSVA and RSVB from the parent virus, and a new mutation in RSVB suggests these viruses are still diverging. Our work suggests that the most effective sterilizing antiviral immunity leads to more rapid virus mutation and immune evasion that applies to RSV treatment, vaccines, and surveillance.
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