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
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ABSTRACT |
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E. Duizer and K. J. Schwab contributed equally to this work.
Present address: Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
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INTRODUCTION |
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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.
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METHODS |
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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 37 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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RESULTS |
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 2 July 2003;
accepted 10 September 2003.