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Volume 13, Number 3—March 2007

Research

In Vitro Cell Culture Infectivity Assay for Human Noroviruses

Timothy M. Straub*Comments to Author , Kerstin Höner zu Bentrup†, Patricia Orosz Coghlan‡, Alice Dohnalkova*, Brooke K. Mayer*1, Rachel A. Bartholomew*, Catherine O. Valdez*, Cynthia J. Bruckner-Lea*, Charles P. Gerba‡, Morteza A. Abbaszadegan§, and Cheryl A. Nickerson†1
Author affiliations: *Pacific Northwest National Laboratory, Richland, Washington, USA; †Tulane University School of Medicine, New Orleans, Louisiana, USA; ‡University of Arizona, Tucson, Arizona, USA; §Arizona State University, Tempe, Arizona, USA;

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Table 1

Summary of methods from the 4 norovius infectivity trials*

Infectivity trial (date)Virus stocks/comments†Time points assayedAssays performed
First (Mar 2005)
Combined equal volumes of strains 149, 155, and flag2 (P0). Effective dilutions of 10–1 to 10–3 were assayed. Supernates were harvested from all infected wells, dilutions of viral stock, and time points combined (≈15 mL final volume).
1 h–72 h; media changed in all wells at 24 h postinfection.
CPE, RT-PCR (Table 3), thin section light microscopy and ultrathin section TEM (Figure 1).
Second (Jun 2005)
Supernate cocktail from first infectivity trial (P1), strains 149 and flag2 tested alone (P0 stool samples). Supernates from each time point were harvested for subsequent infectivity trials (≈3 mL/time point).
Same as first infectivity trial; media changed every 24 h.
Same as first infectivity trial, except that CPE (Figure 2) was documented photographically (Figure 3 refers to TEM).
Third (Aug 2005)
Supernate from combined stock (P2), 149, and flag2 (P1), stool sample flag2 (P0) were harvested. Controls generated by ultrafiltration (10,000 MWCO).
Same as second infectivity trial.
Same as second infectivity trial (Figure 4); first attempt with FISH
Fourth (Dec 2005)Infectivity followed through 5 passages in cell culture using strains 155 and flag2 (P0–P5). Effective dilution of viruses at P5 = 1:106, if replication was not occurring.Infected aggregates were processed at 24 h postinfection, and the viruses were used for subsequent passage.CPE, RT-PCR, molecular beacon FISH (Figure 5). PCR products from P3 of both strains were cloned and sequenced. Compared sequences with sequenced PCRs from original stools.

*CPE, cytopathic effect; RT-PCR, reverse transcription–PCR; TEM, transmission electron microscopy; MWCO, molecular weight cutoff; FISH, fluorescence in situ hybridization.
†Passage no. (P#) definitions: P0, viruses from a stool sample and used to infect a cell culture for the first time; P1, viruses harvested from P0 cell cultures and used to infect cell culture a second time; P2, viruses harvested from P1 and used to infect cell culture a third time; P3, viruses harvested from P2 and used to infect cell culture a fourth time; P4, viruses harvested from P3 and used to infect cell culture a fifth time; and P5, viruses harvested from P4 and used to infect cell culture a sixth time.

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1Current affiliation: Arizona State University, Tempe, Arizona, USA

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