Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation

Human parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses. IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions

Isolation and characterization of the fall Chinook aquareovirus

Abstract Background Salmon are paramount to the economy, ecology, and history of the Pacific Northwest. Viruses constitute one of the major threats to salmon health and well-being, with more than twenty known virus species that infect salmon. Here, we describe the isolation and characterization of the fall Chinook aquareovirus, a divergent member of the species Aquareovirus B within the family Reoviridae. Methods The virus was first found in 2014 as part of a routine adult broodstock screening program in which kidney and spleen tissue samples from healthy-appearing, adult fall Chinook salmon (Oncorhynchus tshawytscha) returning to a hatchery in Washington State produced cytopathic effects when inoculated onto a Chinook salmon embryo cell line (CHSE-214). The virus was not able to be confirmed by an RT-PCR assay using existing aquareovirus pan-species primers, and instead was identified by metagenomic next-generation sequencing. Metagenomic next-generation sequencing was used to recover the full genome and completed using 3′ RACE. Results The genome of the fall Chinook aquareovirus contains 11 segments of double-stranded RNA totaling 23.3 kb, with each segment flanked by the canonical sequence termini found in the aquareoviruses. Sequence comparisons and a phylogenetic analysis revealed a nucleotide identity of 63.2% in the VP7 gene with the Green River Chinook virus, placing the new isolate in the species Aquareovirus B. A qRT-PCR assay was developed targeting the VP2, which showed rapid growth of the isolate during the initial 5 days in culture using CHSE-214 cells. Conclusions This sequence represents the first complete genome of an Aquareovirus B species. Future studies will be required to understand the potential pathogenicity and epidemiology of the fall Chinook aquareovirus

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

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

Gene editing and elimination of latent herpes simplex virus in vivo

Herpes simplex virus establishes lifelong latency in ganglionic neurons, which are the source for recurrent infection. Here Aubert et al. report a promising antiviral therapy based on gene editing with adeno-associated virus-delivered meganucleases, which leads to a significant reduction in ganglionic HSV loads and HSV reactivation