|DANCHO, BROOKE - Animal And Plant Health Inspection Service (APHIS)|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/15/2012
Publication Date: 7/21/2012
Citation: Kingsley, D.H., Dancho, B. 2012. An extraction method for discrimination between infectious and inactive norovirus using RT-PCR. Meeting Abstract. American Society for Virology., Madison, Wisconsin., July 21-25, 2012., Volume 1, Page 1.
Technical Abstract: Human noroviruses (NoVs) are known to bind to human histo-blood group antigens, as well as to chemically-similar porcine gastric mucins. The binding ability of NoV to porcine mucin was assessed as a novel means of distinguishing non-infectious viral particles from potentially infectious viral particles usingqRT-PCR. Over 68 percent of GI.1 NoV (Norwalk strain) was observed to bind to porcine gastric mucin-conjugated magnetic beads (PGM-MBs). After thermal treatment, we found decreased binding to PGM-MBs when samples were heated from 0 to 80 deg C. Ultraviolet treatments of 0.5 and 2 J/sqcm reduced observed PGM-MB binding of norovirus to 33 and 0%, respectively, from an initially observed 84 % binding for untreated NoV. A previously reported high pressure processing study (Leon et al., 2011) has, for the first time, defined conditions for inactivation of GI.1 NoV, determining that 4-log of virus was non-infectious to human volunteers after a 5-min, 600-MPa pressure treatment at 6 deg C. Similar 600-MPa high pressure treatments reduced binding of the virus to PGM-MBs by 4.7-log, as determined by qRT-PCR, while a 300-MPa pressure treatment, which is not sufficient to inactivate GI.1 NoV, reduced binding to PGM-MBs by only 0.45-log. These results indicate that the loss of NoV binding to porcine mucin can be used as a means to preferentially exclude non-infectious virus particles from subsequent RT-PCR detection and analysis by selective extraction.