Laboratory efforts to cultivate noroviruses

Erwin Duizer1, Kellogg J. Schwab2,{dagger}, Frederick H. Neill2, Robert L. Atmar2, Marion P. G. Koopmans1 and Mary K. Estes2

1 Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institutes for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, The Netherlands
2 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA

Correspondence
Erwin Duizer
Erwin.Duizer{at}rivm.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide and are recognized as the foremost cause of foodborne illness. Despite numerous efforts, routine cell cultures have failed to yield replicating NoV. This paper describes methods used to try to grow NoV in vitro in two laboratories. Cells (A549, AGS, Caco-2, CCD-18, CRFK, CR-PEC, Detroit 551, Detroit 562, FRhK-4, HCT-8, HeLa, HEC, HEp-2, Ht-29, HuTu-80, I-407, IEC-6, IEC-18, Kato-3, L20B, MA104, MDBK, MDCK, RD, TMK, Vero and 293) were cultured on solid or permeable surfaces. Differentiation was induced using cell culture supplements such as insulin, DMSO and butyric acid. In some cases, the cells and the NoV-containing stool samples were treated with bioactive digestive additives. Variables evaluated in cultivation experiments included the method of preparation of the virus inoculum, the genotype of the virus, conditions for maintenance of cell monolayers, additives in the maintenance medium and the method of inoculation of the cells. Serial blind passage studies were performed routinely. In addition to evaluation for CPE, evidence of virus replication was sought using immunofluorescent assays to detect newly produced viral capsid antigen and RT-PCR assays to detect the viral genome. Although some infected cultures remained NoV positive by RT-PCR for up to five passages and an occasional cell in a monolayer showed evidence of specific immunofluorescence, no reproducible NoV-induced CPE was observed and all RT-PCR results that were positive initially were negative following continued passaging. Thus, attempts to develop a method for the cultivation of NoV were unsuccessful.

E. Duizer and K. J. Schwab contributed equally to this work.

{dagger}Present address: Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Acute gastroenteritis is one of the most common infectious diseases of humans. In developed countries, human enteric caliciviruses (HECVs) are recognized as the major causative agents of community cases and outbreaks of acute non-bacterial gastroenteritis. HECVs are divided into two genera, the genus Norovirus (NoV, formerly known as ‘Norwalk-like viruses’) and the genus Sapovirus (SaV, formerly known as ‘Sapporo-like viruses’). Recent studies have determined that NoVs are among the leading causes of food- and waterborne gastroenteritis in Western countries, such as The Netherlands, the UK and the USA (de Wit et al., 2001; Hale et al., 2000; Fankhauser et al., 2002). The genus Norovirus is currently subdivided into two genogroups, GGI and GGII, on the basis of sequence homologies of the capsid gene RNA, although subdivision into five genogroups has been proposed recently (Karst et al., 2003).

The study of these highly contagious viruses has been hampered by the lack of in vitro cultivation methods or animal models. Nonetheless, since their discovery in 1972 by immunoelectron microscopy (Kapikian et al., 1972), a vast amount of information has been gathered on the aetiology, the (molecular) epidemiology and the structure of HECVs (reviewed by Atmar & Estes, 2001; Koopmans et al., 2002, 2003). HECV-related gastroenteritis is a self-limited disease and is characterized by diarrhoea, vomiting and malaise (Rockx et al., 2002). Biopsies taken from experimentally infected volunteers showed histopathology in the proximal small intestine that consisted of villous shortening, crypt hypertrophy and mucosal inflammation on the tissue level and shortened microvilli and intracellular vacuolization on the cellular level (Schreiber et al., 1973; Dolin et al., 1975). Although not conclusive, these data suggest that in humans, virus replicates in the proximal small intestine (duodenum and jejunum). Experiments with recombinant Norwalk virus (NV) particles and human gastrointestinal biopsies showed preferential binding to epithelial cells of the pyloric region of the stomach and to enterocytes on duodenal villi. Attachment to the cells was found to correlate with H type 1 antigen expression and occurred only to cells from histo-blood group antigen-secreting individuals (Marionneau et al., 2002). Most routine cell cultures lack the characteristics of these specialized human intestinal epithelial cells in vivo. However, significant attachment and entry of recombinant NV-like particles to differentiated Caco-2 cells was shown (White et al., 1996). Differentiated Caco-2 cells resemble mature enterocytes, express the H antigen and were derived from an individual with blood type O (Amano & Oshima, 1999).

We hypothesized that successful replication of HECV in vitro depends on our ability to mimic the exact stage of differentiation of the intestinal epithelial cells and perhaps even to match the (luminal) microenvironment in a cell culture system. In this manuscript, we describe the diverse efforts undertaken by two laboratories (Houston, TX, USA, and Bilthoven, The Netherlands) to develop a cell culture method for these viruses. Many of our efforts were modelled after methods known to be successful for cultivating other fastidious enteric viruses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell Cultures.
The origin and properties of the cell lines used in this study are listed in Table 1. All cells, except TMK cells, were obtained either directly or indirectly from the ATCC and were routinely grown in standard culture medium (SCM, as specified by the provider). TMK cells were obtained as described (van Wezel et al., 1984). Routine cell cultures were performed in 6-, 12-, 24- or 96-well tissue culture plates and were maintained at 37 °C in an atmosphere of 5 % CO2 and >90 % relative humidity. Modifications to the general cell culture protocol were evaluated and are listed in Table 2. The methods and chemicals used have been associated previously with phenotypic alterations in intestinal epithelial cells that might allow virus cultivation.


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Table 1. Cell lines used in this study

 

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Table 2. Cell phenotype-modifying treatments tested for NoV replication

 
Preparation of inocula
Faecal suspensions.
The inocula used at the RIVM were prepared from stool samples acquired from patients with acute gastroenteritis and who tested positive for HECVs by RT-PCR (Vinje et al., 1997). NoVs were typed and the strains used in this study are listed in Table 3. The original stool samples were either fresh or stored for less than 8 months at 4 °C and tested negative for rotavirus, adenovirus (ELISA, Rotaclone and Adenoclone, Meridian Diagnostics), astrovirus (ELISA, IDEIA, Dako) and enteroviruses (cell cultures on HEp-2, RD and L20B cells). A 10 % (v/v) stool suspension was prepared in DMEM with 100 µg gentamicin ml-1. This suspension was centrifuged for 30 min at 2000 g and subsequently filtered (0·22 µm filter), aliquoted and stored for up to 6 months at -70 °C.


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Table 3. NoV strains used in this study

NoV strains described are classified according to Koopmans et al. (2003).

 
NV inocula.
Stool samples from a large volunteer study completed at Baylor served as the source of NV (Graham et al., 1994). Stool samples from this study were used to obtain the original cDNAs used for sequencing NV (Jiang et al., 1993; Hardy & Estes, 1996). A total of 25 stool samples from 16 subjects were used in our studies. Stool samples were assayed by RT-PCR end-point dilution (denoted as RT-PCR units) to determine the amount of viral RNA present in the sample. One RT-PCR unit is equivalent to approximately 30–40 genomic copies of NV RNA (Schwab et al., 1997). Also, some samples were evaluated for the presence of NV using electron microscopy. In general, samples with the greatest amounts of viral RNA (>105 RT-PCR units ml-1) were used for cell culture studies. Several different methods were used to prepare stool samples for use in cultivation studies. Unformed or watery stools were diluted to 10 % suspensions in PBS, homogenized (three 20 s high-speed bursts on an Omni homogenizer) and clarified for 10 min at 1000 g before being treated with 100 µg gentamicin ml-1 and 10 µg fungizone ml-1 (amphotericin B). Samples were then divided into 1 ml aliquots and stored at -80 °C.

Because problems of cytotoxicity were identified with some of the inocula mentioned above, additional purification steps were included. In one set of experiments, the inoculum was concentrated prior to use by centrifugation through a 30 % sucrose cushion for 3 h at 26 000 r.p.m. in an SW28 rotor (Beckman). In a separate series of experiments, the stool suspension was combined with an equal volume of Freon and homogenized as before. Then, the phases were separated for 10 min at 2000 g. The resulting virus-containing, aqueous supernatant was extracted with fresh Freon in the same manner. The interfaces from these extractions were pooled and re-extracted twice with fresh PBS and the aqueous phases from all of the extractions were combined. To avoid loss of virus, the same blenders and centrifuge tubes were used in these repetitive steps. Virus was pelleted through a sucrose cushion in the same manner as before and the pellet was suspended in 5 ml PBS, resulting in an approximately fourfold concentration of the sample compared to the original 10 % suspension. A subset of these purified virus stocks was further purified and concentrated on a caesium chloride gradient. Caesium chloride was removed by pelleting the virus in a large volume of PBS.

Virus inoculation onto cells
Method.
Prior to inoculation, cells were rinsed with serum-free SCM. The inocula of the first passage consisted of virus suspensions of differing purity and titres of HECV, depending on how the faecal suspensions were purified and concentrated. Based on RT-PCR end-point assays, the seeded virus titres ranged from 102 to 106 PCR units per cell culture well or flask. The inocula were left on the cells or were removed after 1 h and replaced with fresh serum-free SCM. The cells were examined daily for CPE and frozen after 3–7 days. Inocula for the following passages consisted of cell suspensions after one cycle of freeze-thawing, with subsequent treatments as those for the faecal suspensions. Modifications to the general approach were evaluated and are listed in Table 4; the methods used have been associated previously with more efficient or increased virus cultivation.


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Table 4. Variety of methods tested for NoV replication

 
Additives.
Artificial intestinal juices were produced as described by Oomen et al. (2003). Human small intestinal contents were obtained from the duodenum of a patient with an ileostomy and were provided by D. Graham at Baylor College of Medicine (Houston, TX, USA). These intestinal content preparation (ICP) samples were filtered and processed as described by Flynn & Saif (1988). At the RIVM, monkey ICP was obtained from the duodenum of a cynomolgus monkey (28-month-old male), diluted 1 : 1 in PBS, sterilized by filtration (0·22 µm filter) and stored at -20 °C. Additional additives evaluated are listed in Table 5.


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Table 5. Faecal suspension preparations and additives tested for NoV replication

 
Detection of virus replication.
Several methods were used to measure in vitro virus replication. Every cell culture assay was evaluated daily for the presence of CPE in test samples compared to a series of control samples (which were essential to eliminate false-positive results due to sample and/or reagent toxicity to the cells). These controls included NoV-negative stool samples, buffer controls and media additive controls (e.g. intestinal content additives, trypsin, etc.). Virus replication was also monitored by RT-PCR using primers specific for detecting the polymerase region of the NoV genome (Vinje et al., 1997; Schwab et al., 1997). To recover any viral nucleic acid present in the cells (if viruses were replicating), viral RNA was extracted using TRIzol reagent (Gibco-BRL), according to manufacturer's instructions, or by a general phenol/chloroform extraction method (Sair et al., 2002), prior to RT-PCR amplification. Virus replication was also monitored by immunofluoresence (IF) assay. For the IF assay, hyperimmune NV IgG was purified from serum obtained from recombinant NV-immunized rabbits (Graham et al., 1994) using the caprylic acid protocol (McKinney & Parkinson, 1987). Purified anti-NV IgG was labelled with FITC using a FITC-labelling kit (Roche), following the manufacturer's instructions. For immunofluorescent detection of NV on 96-well plates, the culture medium was decanted and the cells were fixed with methanol for 15 min. Following air-drying, the plates were either stored at -20 °C with desiccant or assayed immediately. Sf9 insect cells infected with a recombinant baculovirus that expressed the NV capsid protein, the SaV capsid protein or the rotavirus SA11 VP6 capsid protein were used as positive and negative controls. Infected cells were fixed with methanol and stored at -20 °C until use as controls in IF assays. After rehydration with PBS for 15 min, PBS was decanted and 100 µl of a 1 : 50 dilution of FITC-labelled NV IgG was added to each well and the samples were incubated for 3 h at 37 °C. Cells were subsequently washed three times with 100 µl PBS and evaluated for fluorescence using an inverted fluorescence microscope with an absorbance/emission cube specific for FITC.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Survey of cell cultures for ability to support growth of NoVs
Cell monolayers (Table 1) were monitored for CPE and cell suspensions were analysed by RT-PCR. Most cell suspensions were negative for NoV RNA at passage 5, except for some Caco-2, HuTu-80 and HCT-8 cultures, which were found to remain positive for up to six passages but were negative by passage 7. The virus titre in the inocula used for these assays was sufficiently high to allow the detection of virus inoculum RNA in the diluted samples. Thus, the NoV RNAs detected were thought to represent virus from the inoculum. CPE occurred twice in Caco-2 cell layers, once due to an adenovirus (first detected in the third passage but this was not characterized further) and once due to echovirus type 14 (first detected in the second passage). In a single case, CPE was detected in a butyrate-treated cell layer of Caco-2; this occurred at the same time as the one mentioned in the routine cell cultures and was due to adenovirus. Data on PCR analysis of butyrate (or otherwise) differentiated cells (Table 2) were not different from the routine cell cultures, i.e. NoV was never detected beyond the sixth passage. No correlation was found between the presence of NoV RNA at passage 5 and the genotype of the virus.

NV RNA was detected by RT-PCR in passage 3 harvests following serial passage in I-407 cells. Again, this RNA was thought to represent virus from the inoculum. No NV RNA was detected from passage 5 harvests of AGS, I-407 or Vero cells. Detroit 551 cells demonstrated no evidence of CPE or RNA by RT-PCR following passage 2 (the highest passage number examined for this cell line). None of the methods described in Table 2 produced evidence of NoV replication.

Effect of different treatments of virus inoculum or cultured cells on NoV replication
Extensive studies were performed using three, high-titred NV inocula and duodenal or ileal filtrate on Caco-2 and HEC cells. No replication of virus was detected by CPE, RT-PCR or IF assay following seven passages using the duodenal filtrate. Punctate fluorescence in multiple Caco-2 cells was seen in one well after ten passages using cells supplemented with the ileal filtrate. Throughout these studies, individual cells that reacted with the NV-specific antibody were detected by IF assay. These fluorescent-positive cells tended to be found on top of the cell monolayer, were few in number and were not found in control monolayers. However, no signal was detected following passage of harvested material from these cells or their supernatants. In addition, viral RNA was not detected following serial passage of material from these cultures. Fluorescent-positive cells were seen with several different inocula and in a number of different cells lines (data not shown). However, NV RNA could not be detected by RT-PCR and no evidence of virus replication was detected by IF assay following passages 11 and 12. None of the methods described in Table 4 resulted in virus-induced CPE and no NoV RNA was detected after five passages.

Intestinal enzymes and ICPs were tested to determine if any of these treatments would support NoV replication in the cell lines used (Table 5). Morphological analyses for CPE were complicated by the toxic effect of the additives. Cell monolayers exposed to gastric juice, duodenal juice and trypsin (>1 µg ml-1) exhibited areas of detached cells and spots with rounded or pyknotic cells. No virus-dependent CPE was detected in any of the cultures (Table 5) and none of the cell suspensions of the fifth passage were PCR positive for NoV RNA.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We systematically evaluated a variety of cell lines and laboratory methods in an effort to replicate NoVs. Based upon the current understanding of the binding and replication sites of NoV in vivo (Dolin et al., 1975; Marionneau et al., 2002), we created in vitro cell culture systems that mimic the epithelium of these sites using gastric cells (AGS and Kato-3), duodenal cells (HuTu-80) and small intestinal enterocyte-like cells (Caco-2) and allowing them to differentiate. None of the cell culture combinations was successful in producing in vitro NoV replication, as determined by multiple assays. Because all attempts were unsuccessful, it is difficult to determine which steps are critical for NoV replication. However, based on the number of unsuccessful attempts, it might be reasoned that at least one, and probably several, steps in the replication cycle are very critical, i.e. require specific features from host cells, the virus or both.

The addition of a variety of additives, including proteases, hormones and intestinal contents, did not lead to successful NoV cultivation, although this approach has enhanced the growth of other caliciviruses; the addition of trypsin enhances the growth of canine calicivirus (Schaffer et al., 1985), and porcine enteric calicivirus (Cowden strain) was adapted to cell culture in a porcine kidney cell line after inclusion of ICP from gnotobiotic pigs (Flynn & Saif, 1988; Parwani et al., 1991). The nature of the necessary factor in the ICP has not yet been elucidated but it may affect replication by triggering a cellular cAMP-signalling pathway (Chang et al., 2002). Despite adaptation of the virus to in vitro propagation, involving several mutations in the protease, RNA polymerase and capsid protein genes (Guo et al., 1999), the inclusion of ICP is still necessary. The human ICPs used in our studies were collected from adult subjects, and if virus-specific antibodies were present in the specimens the virus inoculum may have been neutralized. Additional studies with antibody-depleted intestinal contents are warranted.

In our studies, we evaluated a number of different cell lines derived from human and non-human hosts. None of these could be shown to support the in vitro replication of NoVs. In addition, we attempted co-cultivation experiments using fibroblasts and epithelial cells in case the former might provide a critical factor for the differentiation of the latter to support NoV growth. This strategy might still be pursued as better models of in vitro intestinal tissue cultures are developed.

Cell binding is a critical initial step in the virus replication cycle. Recent data have shown the importance of ABO blood groups and secretor status in binding of different genotypes of NoV to epithelial cells (Hutson et al., 2002, 2003; Marionneau et al., 2002). Caco-2 cells are derived from a secretor individual with blood type O and the expression of the relevant H type 1 blood group antigen increases during the enterocytic differentiation of Caco-2 cells (Amano & Oshima, 1999). Moreover, previous studies showed that NV-like particles bind well to differentiated Caco-2 cells, suggesting that the block to replication in these cells is at a post-binding step (White et al., 1996). In addition, soluble capsid protein, which could have interfered with virus binding, was removed in several experiments by pelleting the virus through a sucrose cushion. To circumvent the receptor binding, internalization and uncoating steps, others have tried transfection of cultured cells with purified caliciviral RNA without success (K. Green, NIH, USA, personal communication).

Our studies and previous studies have shown that NoVs are stable viruses (Schwab et al., 2000), so inactivation of the virus is an unlikely mechanism that prevents in vitro cultivation. Viral RNA persisted in cell culture after three passages (>12 days) in the presence of digestive juices. Although the presence of RNA does not necessarily indicate the presence of infectious virus, it does indicate that viral RNA is, in some way, protected from the abundant cellular RNase activity. It has been shown that the virus may persist in the environment for many days and still lead to infection (Schwab et al., 2000). Furthermore, the methods used allowed the detection of other enteric viruses (adenoviruses and enterovirus), indicating that the cells were sensitive to viruses, that stool samples from outbreaks can contain other viruses and that the handling of the stool samples did not lead to the inactivation of other enteric viruses.

In conclusion, all attempts to develop a method for the cultivation of NoV presented here were unsuccessful and a cultivable NoV did not emerge from any of the stool samples tested. However, we have written this publication to provide an overview of what we have tried already to avoid duplication of future laboratory efforts. We consider this valuable data, since the need for a method to culture NoV and to demonstrate its viability has been increasing over the last few years. More attempts are needed using novel approaches and building on current experience. It is hoped that the present report will orient the thinking of other investigators to new and different approaches.


   ACKNOWLEDGEMENTS
 
This work was supported by The Netherlands Centre Alternatives to Animal Use (PAD 97-31), The Netherlands Organization for Scientific Research (NWO 01412028) and in the USA by grants from the National Oceanic and Atmospheric Administration (NA77FD0080) and the National Institutes of Health (DK-58955 and DK56338). We are grateful for the excellent assistance of Astrid de Groot and Barry Rockx.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 2 July 2003; accepted 10 September 2003.