Cell culture assay for human noroviruses.

To the Editor: We read with great interest the article on human norovirus (hNoV) by Straub et al. (1). By using 3-dimensional aggregates of a highly differentiated intestinal epithelial cell line, the investigators claimed to have established an in vitro cell culture model that "support[s] the natural growth of human noroviruses." While the authors provide compelling evidence of successful virus infection through microscopy, hybridization of viral RNA after 5 passages in cell culture, and preliminary evidence of viral RNA replication through limiting dilution PCR, we question the level of virus replication that is actually achieved in this system.

Straub et al. demonstrate through fluorescent in situ hybridization the presence of viral RNA through 5 passages in his system. This phenomenon could be similar to the findings of Duizer et al. (2), if the level of replication simply maintained the viral titer. Therefore, we argue that virus replication curve, estimated by using quantitative real-time PCR or semiquantitative endpoint dilution PCR with the end-dilution of each sample from different time points in this system, will conclusively determine the suitability of this model as a productive virus replication system. To support our hypothesis, we point to the pig model for hNoV infectivity (3). In that study investigators failed to observe an increase in viral shedding from symptomatic piglets upon serial passage, despite successful intracellular detection of viral RNA and newly synthesized virus-encoded protein in host cells dying of apoptosis. This suggests that the demonstration of cytopathic effect and virus internalization in cells alone may not provide direct evidence of productive virus replication. In conclusion, although we acknowledge that Straub et al. have provided evidence of successful hNoV infection in vitro, we suggest subsequent studies to characterize the level of virus replication in this system.

Martin C.W. Chan, * Y.P. Wong, * and Wai K. Leung *

* The Chinese University of Hong Kong, Hong Kong Special Administrative Region, People's Republic of China

References

(1.) Straub TM, Honer zu Bentrup K, Orosz-Coghlan P, Dohnalkova A, Mayer BK, Batholomew RA, et al. In vitro cell culture assay for human noroviruses. Emerg Infect Dis. 2007;13:396-403. Available from http://www.cdc.gov/eid/content/13/3/396.htm

(2.) Duizer E, Schwab KJ, Neill FH, Atmar RL, Knopmans MP, Estes MK. Laboratory efforts to cultivate noroviruses. J Gen Virol. 2004;85:79-87.

(3.) Cheetham S, Souza M, Meulia T, Grimes S, Han MG, Saif LJ. Pathogenesis of a genogroup II human norovirus in gnotobiotic pigs. J Virol. 2006;80:10372-81.

Address for correspondence: Wai K. Leung, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, People's Republic of China; email: [e-mail]

In Response: We appreciate the comments provided by Chan et al., in response to our recently published article (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human norovirus (hNoV) to enhance risk assessments when these viruses are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne hNoV monitoring. However, these assays cannot distinguish infectious from noninfectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will improve risk assessment models and protect human health, regardless of whether we are propagating hNoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply.

Nonetheless, Leung et al.'s assertion regarding the suitability of our method for the in vitro propagation of high titers of hNoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are finding increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools.

Timothy M. Straub, * Kerstin Honer zu Bentrup, ([dagger]) Patricia Orosz-Coghlan, ([double dagger]) Alice Dohnalkova, * Brooke K. Mayer, * (1) Rachel A. Bartholomew, * Catherine O. Valdez, * Cynthia J. Bruckner-Lea, * Charles P. Gerba, ([double dagger]) Morteza A. Abbaszadegan, ([section]) and Cheryl A. Nickerson ([dagger]) (1)

* Pacific Northwest National Laboratory, Richland, Washington, USA; ([dagger]) Tulane University School of Medicine, New Orleans, Louisiana, USA; and ([double dagger]) University of Arizona, Tucson, Arizona, USA

Address for correspondence: Timothy M. Straub, Pacific Northwest National Laboratory--Chemical and Biologic Sciences, PO Box 999, MS P7-50, Richland, WA 99352, USA; email: [e-mail]

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

References

(1.) Straub TM, Honer zu Bentrup K, Orosz-Coghlan P, Dohnalkova A, Mayer BK, Batholomew RA, et al. In vitro cell culture assay for human noroviruses. Emerg Infect Dis. 2007;13:396-403. Available from http://www.cdc.gov/eid/content/13/3/396. htm

(2.) Rochelle PA, Marshall MM, Mead JR, Johnson AM, Korich DG, Rosen JS, et al. Comparison of in vitro cell culture and mouse assay for measuring infectivity of Cryptosporidium parvum. Appl Environ Microbiol. 2002;68:3809-17.