Human parainfluenza virus evolution during lung infection of immunocompromised individuals promotes viral persistence

Abstract

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment.Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We show that intra-host evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favor viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from two immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process.Fixation of this mutation was associated with exposure to a drug that cleaves host cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

Modeling Infection and Tropism of Human Parainfluenza Virus Type 3 in Ferrets

“…Though useful, these AO can be further optimized to perform combined in vitro and in vivo studies about HPIV-3 infectivity and to assess intervention strategies. The finding that primary ferret respiratory epithelial cells are less susceptible and permissive for rHPIV-3 CI-1-EGFP infection and replication than human respiratory epithelial cells is in line with our understanding of HPIV3’s fine specificity for specific host environments ( 39 – 42 , 44 , 45 ). While neonatal ferrets had been shown to be infectible by HPIV-3 that was almost certainly laboratory adapted ( 51 , 52 ), we now show that clinical, nonadapted HPIV-3 strains infect ferrets in vivo .…”

“…The generation of the rHPIV-3 clinical isolate 1 (CI-1)-EGFP (rHPIV-3 CI-1-EGFP) was previously described ( 40 , 41 ). Virus stocks were grown on either HAE at ALI or AO at ALI by inoculating cells with 500 to 5,000 TCID 50 and harvesting in Dulbecco’s phosphate-buffered saline (DPBS) supplemented with Ca and Mg (0.9 mM MgCl 2 and 0.49 mM CaCl 2 ) at 2 to 4 dpi.…”

“…Mechanisms that affect HPIV-3–host cell interplay are finely tuned to the host environment, and clinical isolates differ significantly from laboratory viruses in terms of receptor interaction and viral fusion properties that govern entry and fitness in vivo ( 39 – 42 ). Growing HPIV-3 in standard cell culture leads to immediate adaptations in the fusion/entry complex and the characteristics that confer viral fitness.…”

“…However, in human airway epithelial (HAE) cells and human lung organoid models (airway organoids [AO]), clinical strains of HPIV-3 grow and spread without adaptation ( 43 ). Such human airway models permit the study of HPIV-3 tropism and pathogenesis in the human respiratory epithelium in the absence of an adaptive immune response ( 39 – 45 ). In ex vivo human lung tissue, HPIV-3 primarily infects ciliated epithelial cells and type II pneumocytes ( 43 , 46 ).…”

“…inoculation with different concentrations of virus. This study establishes an infection model for this important pediatric pathogen and lays the groundwork for further prevention and treatment studies in a model that complements human ex vivo lung models and existing HPIV-3 animal models ( 40 – 43 ).…”