1 Department of Pediatrics, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Division of Infectious Diseases, The Women and Children's Hospital of Buffalo, Buffalo, NY 14222, USA
2 Division of Immunologic and Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA
Correspondence
Marie Riepenhoff-Talty
mrtalty64{at}comcast.net
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ABSTRACT |
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INTRODUCTION |
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An evaluation of biological differences between these rhesus and bovine reassortant viruses may be useful. Certain strains of rotavirus of serotype G3, particularly RRV, but also SA11 and HCR3, cause hepatitis and bile-duct obstruction in suckling mice (Petersen et al., 1997, 1998
; Riepenhoff-Talty et al., 1993
; Uhnoo et al., 1990
). WC3 has been shown not to infect liver cells in the same mouse model (Uhnoo et al., 1990
). To date, no studies have been done to determine if the hepatopathogenic potential of RRV, SA11 or HCR3 is maintained when these wild-type G3 rotaviruses are reassorted.
In the present study, we investigated whether Rotashield and WC3-PV would produce infection and disease in infant CB17scid mice. We compared the effects of oral inoculation of these two vaccines by examining clinical, morphological, histopathological and virological parameters after inoculation.
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METHODS |
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Virus and inoculation.
Inocula were provided and coded by Merck Research Laboratories, West Pt, PA, USA (Rotashield was from vials purchased over the counter). The code was broken after completion of data analysis.
A lyophilized preparation of Rotashield contained one rhesus strain, RRV-G3, and three rhesushuman reassortant rotaviruses. Each reassortant rotavirus contained 10 gene segments from RRV and one gene (segment 9) from human rotavirus, which represented serotypes G1, G2 or G4. This preparation, RRV-TV, was reconstituted in 1 ml Eagle's minimal essential medium (EMEM), and 1-day-old pups were inoculated orally with 20 µl containing 8x103 p.f.u. (2x103 p.f.u. of each strain) of virus. WC3-PV contained five bovinehuman reassortants, and is identical to RotaTeq in terms of the virus reassortants included and their concentration. RotaTeq differs only in that the virus is suspended in a different buffer/stabilizing diluent. Each bovinehuman reassortant rotavirus contained gene segments from bovine strain WC3 and human gene segment 4 or 9 (derived from human rotaviruses representing serotypes P1a, G1, G2, G3 or G4). Reassortants G1 and G2 also contain gene 3 from a human G1 rotavirus (strain WI79) (H. F. Clark and others, unpublished results). One-day-old pups were inoculated orally with 20 µl containing 5x105 p.f.u. WC3-PV (1·0x105 p.f.u. per strain) diluted in Williams' E medium (Hyclone) containing 12·5 µg trypsin ml1 (Gibco-BRL). Each dose represented 2 % of the quantity of infectious virus administered in studies of human infants. One-day-old control group pups were inoculated with 20 µl of medium alone.
Clinical disease follow-up and diagnostic criteria.
Animals were observed daily for signs of diarrhoea and hepatitis. The clinical diagnostic criteria for diarrhoea were watery or soft stools with mucus. The diagnostic criteria for hepatitis were established based on evidence from our previous studies (Riepenhoff-Talty et al., 1993; Uhnoo et al., 1990
). Pups were diagnosed with hepatitis when they met two or more of the following criteria: (i) clinical signs including specific findings, e.g. jaundice with or without white- or clay-coloured faeces, and non-specific findings, e.g. malnourished appearance, lethargy and greasy fur; (ii) virus isolation was positive for rotavirus in the liver tissue; (iii) rotavirus antigen was detected in the liver tissue; (iv) gross liver appearance was abnormal.
Animal dissection/specimen collection.
Pups were sacrificed according to a dissection schedule, approximately every other day post-inoculation up to 23 days. Animals exhibiting signs of disease were generally sacrificed at the onset of these signs. Pups were anaesthetized with Halothane (Halocarbon Laboratories). Cardiac blood was collected and, for pups at or less than 5 days post-inoculation (p.i.), whole blood was suspended in EMEM. For those pups sacrificed at day 7 p.i. or after, blood was centrifuged, serum and cell pellets were separated and both were suspended in EMEM. All blood samples were kept at 70 °C for further use. Gross examination of liver, bile duct, gall bladder, spleen and intestine was carried out before sampling. The liver was examined for evidence of macroscopic changes such as areas of necrosis, grey or white patches and abnormal colours (grey, yellow, dark-bile). Pups were also examined for bile-duct obstruction, defined as an enlarged gall bladder engorged with dark bile.
Tissue samples of intestine, spleen and liver were removed and distributed as follows: in 0·5 ml EMEM for virus isolation; embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen in isopentane/liquid nitrogen for immunofluorescent antibody (IFA) assay; in 0·5 ml 10 % formalin for haematoxylin and eosin (H&E) histochemical staining and immunoperoxidase antibody (IPA) assay. In order to ensure consistency of sampling, the samples were taken and processed as follows: the first 0·5 cm of duodenal tissue was taken from the proximal point and placed in EMEM, the second 0·5 cm of adjacent tissue was placed in O.C.T. and the third 0·5 cm in formalin. For the jejunal biopsy, 1 cm of the mid-portion was collected for H&E staining and IPA assay, the right 1 cm for IFA assay, and the left 1 cm for virus isolation; for the ileal biopsy, 1 cm of the ileum was taken for virus isolation from the distal point, the adjacent 1 cm for IFA assay and the next 1 cm for H&E staining and IPA assay. The ilealcaecal junction, colon contents and pancreas (if found) were also collected for virus isolation. The spleen was measured and cut into three equal parts for virus isolation, IFA plus IPA assays and H&E staining. Samples of liver tissue were taken as follows: the left lobe was cut into three pieces for virus isolation, IFA plus IPA assays and H&E staining; all other lobes were collected for virus isolation. All samples in O.C.T. and EMEM were stored at 70 °C until further use. All tissue samples for virus isolation were weighed before suspension in EMEM in order that virus titres could be determined on the basis of p.f.u. (g tissue)1. Samples in formalin were embedded in paraffin on the day of collection. To avoid contamination, all dissection instruments were sterilized prior to use and a different set was used for each pup. The instruments used for one pup were disinfected in ethanol between each tissue sampling.
IPA assay.
Tissues (intestine, liver, spleen) fixed in formalin were embedded in paraffin, sectioned and subsequently assayed for rotavirus antigen. The primary antibody (rabbit anti-human rotavirus antibody) was purchased from Dako. The detection procedure using rabbit polyclonal antibody and an HRP detection system was done according to the supplier's instructions (Signet Laboratories). Positive and negative controls were included for each assay. Sections were scored as either positive or negative when compared with the controls.
IFA assays.
Sections of liver (5 µm) were used for IFA assay. Tissue sections were fixed in cold (4 °C) acetone for 10 min. After rehydration in PBS, 10 µl of rabbit anti-rotavirus antibody (Dako) diluted 1 : 10 in PBS was added and the slides were incubated overnight at 4 °C. After washing the slides three times in PBS, 10 µl fluorescein-conjugated swine anti-rabbit antibody (Dako) diluted 1 : 20 in 0·005 % Evans' blue was added to the tissue. Slides were incubated at 37 °C for 30 min. After further washes, the slides were air-dried and covered with PBS/glycerol and a cover-slip. Slides were examined with a BH-2 Olympus microscope equipped with a mercury vapour bulb. Positive and negative controls were included for each assay and slides were read as positive (appearance of specific apple-green fluorescence) or negative (lack of fluorescence) compared with controls.
Virus isolation and identification.
Tissue samples (including liver, spleen, various intestinal tissues and pancreas) were minced with scissors and then frozen in a dry-ice/ethanol bath and thawed in a 36 °C water-bath. The freezethaw cycles were done three times. Blood-cell clots were resuspended for 1 min before being subjected to three freezethaw cycles. Serum was also subjected to three freezethaw cycles. All samples were then vortexed for 1 min and centrifuged for 30 s at 1100 g. Undiluted supernatant fluid from all the samples was assayed.
Quantities of infectious rotavirus were titrated using a previously described plaque assay (Offit et al., 1983). To avoid bias, every individual plaque that was well separated was harvested from all wells that contained 20 plaques and resuspended in Dulbecco's minimal essential medium containing 0·5 µg trypsin ml1 (crystallized; Sigma) with 20 U penicillin ml1 and 20 µg streptomycin ml1. Individual plaque suspensions were inoculated onto a monolayer of MA104 cells (Whittaker Bioproducts) in a 24-well plate (Falcon, Becton Dickinson). Plates were checked daily until the maximum cytopathic effect was reached. Rotavirus plaques were characterized by PAGE RNA electropherotyping (Dolan et al., 1985
) and compared with positive controls (the inoculum viruses). Samples with a faint banding pattern were subjected to a second passage in MA-104 cells and were reanalysed. Results were considered negative if rotavirus-specific RNA was not detected by PAGE.
Statistics.
Data were analysed by a 2 test. All comparisons were made between the two vaccine groups and did not include the placebo controls.
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RESULTS |
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Hepatitis and bile-duct obstruction
A total of nine mice that received Rotashield had evidence of developing hepatitis (Table 2). However, since data from our previous (Uhnoo et al., 1990
) and present studies have shown an incubation period of approximately 7 days for development of hepatitis or bile-duct obstruction after oral inoculation of RRV, we have chosen as our denominator the number of mice remaining alive (not sacrificed or dead) after day 6 p.i. For any mice sacrificed from days 1 to 6 p.i., the incidence of hepatitis remained unknown. One pup with hepatitis (SH3) was excluded from the numerator because it was not alive after day 6 p.i. Therefore, the incidence rate for Rotashield-induced hepatitis was 62 % (8/13) (P<0·005), compared with none of 11 mice given WC3-PV.
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Hepatobiliary disease associated with Rotashield was not confined to particular litters, because evidence of infection could be found in pups in every cage. However, no cage contained pups that were all equally affected. Further evidence that disease and death were randomly distributed among litters was the finding that three missing pups among the Rotashield-inoculated mice, presumed to have been cannibalized by their mothers after the onset of disease, were each members of different litters. This exclusive association of Rotashield with disease was reinforced by the appearance of disease only in cages containing Rotashield-infected animals.
Mice that received WC3-PV or placebo did not show any signs of developing hepatitis. Although infectious virus was isolated from the livers of mice inoculated with WC3-PV on day 1 p.i. (Table 3), it was not recovered at any later time, and no further evidence in terms of clinical signs, antigen detection, gross organ findings or hepatopathological changes were seen that supported the diagnosis of hepatitis in these mice.
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Recovery of virus from Rotashield-inoculated mice followed a biphasic pattern (Table 4). Each of three mice sacrificed from days 1 to 3 p.i. exhibited low-titre infections, primarily in the gut. From 5 to 9 days p.i., virus was recovered from the intestine of only one of five animals and not from any other tissues. A resurgence of virus was detected throughout the viscera from days 10 to 15 p.i. Virus was recovered from four of five mice during this time period. Particularly high titres (>107 p.f.u. g1) were identified in the liver and pancreas. After day 15 p.i., the rate of infection declined again. In five surviving animals, virus was recovered only from the MLN (105 p.f.u. g1) of pup S4H21 and the liver (2·4x101 p.f.u. g1) of pup SH23.
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DISCUSSION |
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Only RRV-G3 was isolated from samples taken from pups inoculated with Rotashield, despite the fact that the RRV-G1, RRV-G2 and RRV-G4 reassortants were present in equal proportions to RRV-G3 in the vaccine. In a study of oral inoculation of Rotashield into adult BALB/c mice, RRV-G3 was the virus most frequently isolated from samples of Peyer's patches (PP), and predominantly RRV-G3 but also G1 and G2 reassortants were recovered from MLN (Moser et al., 2001).
In separate studies (unpublished), immunocompetent newborn BALB/c mice and gnotobiotic newborn piglets were inoculated with comparable doses of Rotashield (on the basis of p.f.u. g1). In newborn BALB/c mice, virus was rarely recovered from gut, liver and MLN, and was exclusively RRV-G3. In contrast, virus recovered from visceral organs of piglets included RRV-G3, and G2 and G4 reassortants of RRV (L. Saif, M. Riepenhoff-Talty, H. Qiao & H. F. Clark, unpublished data). In clinical trials of Rotashield in infants, RRV predominated but RRV reassortants were also recovered from faeces (Kobaysshi et al., 1994; Ward et al., 1998
). In an investigation of horizontal spread of RRV-TV (Rotashield) viruses into 29 non-inoculated infants in Venezuela, RRV-G3 recovery predominated but several ST3 (rotavirus strain St Thomas 3)xRRV reassortants (G4) were also recognized (Hoshino et al., 2003
).
Since various reassortants of RRV have been recovered from orally inoculated infants, piglets and adult BALB/c mice given Rotashield, but only RRV was recovered from immunocompetent (see above paragraph) or SCID mice fed Rotashield, it appears that absolute selective replication of the RRV component of animals fed Rotashield is unique to the newborn mice. As VP7 is the only protein that varies in Rotashield components, it appears that VP7 is critical in controlling invasion of and replication within liver cells. Alternatively, since only RRV was found in the gut, RRV VP7 may be necessary only to allow replication in the gut and that it is enteric replication that might lead to viraemia. If so, the ability of the RRV VP7 reassortant to replicate in visceral organs could be determined only by parenteral inoculation. If it were determined that parenteral inoculation of Rotashield into newborn mice led to replication of Rotashield reassortant viruses in visceral organs, the observation would be of questionable significance since all natural exposure to rotavirus (as well as administration of rotavirus vaccine) is by oral inoculation. The observation that a selected heterologous hostrotavirus system involving a virus heterologous to that host causes disease (hepatitis and biliary obstruction) not demonstrated in any known natural homologous hostrotavirus system is of great interest.
The suggested critical role of VP7 in the RRVnewborn BALB/c mouse system supports previous observations that, in studies with rotavirus reassortants in pigs, genes 3, 4, 9 and 10 all influence virulence (Hoshino et al., 1995). However, in other studies (Mossel & Ramig, 2002
) on orally inoculated newborn mice, gene 7 (product NSP3) was the predominant determinant of spread of rotavirus to the liver. Factors controlling hepatotropism of rotavirus in the mouse are not yet completely understood.
Although the time of appearance and pattern varied, rotavirus was detected in the bloodstream of mice after oral inoculation with both vaccines. Possible routes for transit of virus to the bloodstream are outlined in Fig. 2(a) (RRV) and (b) (WC3-PV). We suggest that virus may reach the bloodstream and viscera by two common routes: directly into the blood circulation from the intestine or indirectly through the M cells, PP, MLN and spleen. This second route was established by Dharakul et al. (1988)
using murine rotavirus and immunocompetent infant BALB/c mice This pathway would play a diminished role in immunodeficient mice, although uptake by macrophages and interaction with non-specific immune cells would occur. In the current study we did not detect RRV in the bloodstream until day 10 p.i. and this appearance coincided with replication in the liver. It is likely that the small inoculum of RRV and the initiation of the resultant infection in the intestine could account for insufficient inoculum virus reaching the blood circulation. To explain how virus reaches the liver we postulate a third pathway, an ascending route through the bile ducts and hepatic ducts to the liver. There are several lines of evidence that point to an ascending route. First, as mentioned above, the lack of detectable virus circulating in the blood would be a factor. There was also the bimodal nature of virus shedding in the gut, with a distinct lag before liver infection was established. If large amounts of virus were available to the liver cells in the first 2448 h via the blood circulation, one would not expect it to take 710 days for progeny virus to be detected in the liver. Furthermore, bile-duct obstruction appeared to occur at the same time or perhaps earlier than hepatitis. In addition, a previous study that documented the location of the obstruction in the RRV-infected mice showed a consistent pattern, with the lesion at the distal end of the common bile-duct where it empties into the duodenum (Riepenhoff-Talty et al., 1993
).
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We documented the active replication of RRV in hepatocytes by positive findings with IFA and IPA assays. These assays detect intracellular (intracytoplasmic) viral antigen in fixed cells. Virus isolation is more sensitive, and detects fully infectious virions, but does not reveal the location of the virus. It does not distinguish between virus in the blood circulation of an organ or virus replicating in the cells of the organ. However, detection of large deposits of viral antigen in organ tissue is consistent with active virus replication in that organ.
The detection of relatively high titres of RRV in the pancreas of Rotashield-inoculated mice is also intriguing. However, because individual pancreatic samples were insufficient for secondary detection by antigen assay, we could not confirm RRV replication in the pancreas. Three of the four mice with high titres of RRV in the pancreas also had RRV in the blood, but in much lower titres. Follow-up studies are needed to determine whether our observation that mouse pancreas may support RRV replication is real. There is a recent report describing the growth of rotavirus in primary human pancreatic cell culture (Coulson et al., 2002).
Our findings of a low incidence of diarrhoea in the Rotashield-inoculated mice were not totally unexpected. Uhnoo et al. (1990) reported diarrhoea in 80 % of CB17scid pups inoculated with a similar dose (103 f.f.u.) of purified RRV. In the present study, it appears that the ability of RRV-G3 to produce diarrhoea is diminished in Rotashield, possibly by interference mediated by RRV reassortants.
In this study, we elected to keep the Rotashield titre very low to maintain the same ratio of Rotashield titre to WC3-PV titre as used in clinical practice. Preliminary studies using BALB/c mice (the IgH congenic partner of CB17scid mice) showed only minor changes after inoculation of the similar titre of Rotashield (unpublished data). We then examined the more susceptible CB17scid newborn mouse model, which enabled us to maintain the same relative Rotashield/WC3-PV virus titre.
We found clear biological differences between Rotashield and WC3-PV in the suckling CB17scid mouse model. These findings confirm and extend the work of Uhnoo et al. (1990), who studied the WC3 and RRV parental rotavirus strains in the same mouse model. Data from this study again suggest that RRV has unusual and sometimes severe pathogenic potential, and the ability to cross species barriers efficiently. These observations suggest that it might not be the first choice as a vaccine candidate.
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ACKNOWLEDGEMENTS |
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Received 24 September 2003;
accepted 20 April 2004.
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