Production, purification, and capsid stability of rhinovirus C types.
The rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification.
Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, Madison, United States; Cellular & Molecular Pathology Graduate Program, University of Wisconsin, Madison, United States; Medical Scientist Training Program, University of Wisconsin, Madison, United States. Electronic address: tgriggs@wisc.edu.
Theodor F
TF
Griggs
Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, Madison, United States.
Yury A
YA
Bochkov
Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, Madison, United States.
Kazuyuki
K
Nakagome
Institute for Molecular Virology, University of Wisconsin, Madison, United States.
Ann C
AC
Palmenberg
Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin, Madison, United States; Cellular & Molecular Pathology Graduate Program, University of Wisconsin, Madison, United States.
James E
JE
Gern
D002213:Capsid / Q000187:drug effects* / Q000528:radiation effects*; D003902:Detergents / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D050296:Microbial Viability / Q000187:drug effects* / Q000528:radiation effects*; D009997:Osmotic Pressure; D012229:Rhinovirus / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology* / Q000528:radiation effects; D013696:Temperature; D014773:Virology / Q000379:methods*
D003902:Detergents
Centrifugation; Concentration; Purification; Rhinovirus C; Stability