Human Norovirus GII.4 Strain Does Not Replicate in CV-1 Cells or HAV-persistently Infected A549 Cells

Introduction: The successful cultivation of food-borne viruses is a high-priority research outcome with respect to food safety.   An effective cell culture model for noroviruses (NoV), the most common agent of non-bacterial gastroenteritis in humans, has been unattainable despite numerous attempts in many cell lines.  The lack of suitable in vitro culture methods for NoV has lent impetus to efforts in this field, as it would vastly improve food-related surveillance, epidemiology, and trace-back studies. 

Purpose: The objective of this investigation was three-fold: (1) to determine if NoV will replicate in the African green monkey kidney cell line, CV-1, (2) to determine whether Hepatitis A virus (HAV) can function as a helper virus to support NoV replication in A549 cells (human lung carcinoma) persistently infected with HM175/clone 1 strain of HAV, and (3) to determine if incubation of these post-infected (NoV) cells at reduced cell density/reduced temperature can promote NoV cpe/replication, as we have shown previously for persistently HAV-infected A549 and FRhK-4 cells.

Methods: Human stool, positive for NoV, was prepared as 10% clarified suspension in PBS, filtered, and quantified by qRT-PCR, and cell cultures were infected at an “moi” of 30 viral genome copies/cell.  Post-adsorption supernatants were collected immediately after initial infection (2h at 37°C).  Cell pellets and culture supernatants from cells incubated at 37 and 33°C were collected every week up to 7-8 weeks post-infection, pellets extracted for viral RNA, and tested for NoV, HAV, and non-NoV/non-HAV enteric virus replication using RT-qPCR.

Results: Input virus and post-adsorption supernatants tested positive for NoV, while all other samples were negative.

Significance: Reduced temperature and/or persistent (HAV) infection did not function to support NoV virus replication suggesting:  neither cellular nor viral factor(s) in these cell lines, that contribute to the augmentation of HAV replication/cpe under similar incubation conditions, function to support NoV replication.