Department of Molecular Virology & Microbiology, Baylor College of Medicine, One Baylor Plaza, Mailstop BCM-385, Houston, TX 77030, USA1
Veterans Affairs Medical Center, Houston, TX 77030, USA2
Author for correspondence: Margaret Conner at Baylor College of Medicine. Fax +1 713 798 3586. e-mail mconner{at}bcm.tmc.edu
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Abstract |
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Introduction |
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Different constellations of rotavirus genes encoding both structural and non-structural proteins (VP3, VP4, VP7, NSP1, NSP2 and NSP4) are implicated in rotavirus host range restriction (Bridger et al., 1998 ; Broome et al., 1993
; Ciarlet et al., 1998a
; Gombold & Ramig, 1986
; Hoshino et al., 1995
). Among all rotavirus molecular or antigenic features examined, such as RNA electropherotype, subgroup specificity, G serotype or P serotype (genotype), genome segment 5 (encoding NSP1) shows one of the highest degrees of sequence variation among rotavirus strains isolated from humans, pigs, horses, mice, cows, monkeys and cats (Dunn et al., 1994a
; Fujiwara & Nakagomi, 1997
; Hua et al., 1994
; Kojima et al., 1996
; Mitchell & Both, 1990
; Palombo & Bishop, 1994
; Xu et al., 1994
). Although not universal, NSP1 sequence homology of over 40 rotavirus strains shows the best association with species of origin and can best determine interspecies relatedness within the overall genomic RNA constellation (or rotavirus genogroup) (Dunn et al., 1994a
; Fujiwara & Nakagomi, 1997
; Hua et al., 1994
; Kojima et al., 1996
; Nakagomi & Kaga, 1995
; Nakagomi & Nakagomi; 1996
; Palombo & Bishop, 1994
; Wu et al., 1993
). Sequence analyses have confirmed this relatedness or revealed exceptions based on (i) close interspecies relationships between human and animal strains as per RNARNA hybridization, (ii) examples of interspecies transmission of rotavirus strains (human Ro1845, feline FRV-1, equine H-1), or (iii) naturally occurring rotavirus reassortant strains (feline Cat2) between different species or genogroups (Dunn et al., 1994 a
; I
aet al., 1996
; Fujiwara & Nakagomi, 1997
; Kojima et al., 1996
; Nakagomi & Kaga, 1995
; Palombo & Bishop, 1994
; Xu et al., 1994
). A notable exception is that the level of identity of NSP1 between simian rhesus rotavirus (RRV) and SA11 strains is low (57%) (Dunn et al., 1994a
). Because RRV and SA11 are the only two simian rotaviruses isolated to date, it is difficult to speculate why these simian NSP1s are so diverse.
In mice, gene 5 segregates with transmission of virus to uninoculated littermates (Broome et al., 1993 ; Gombold & Ramig, 1986
), but in piglets, gene 5 was not implicated in rotavirus host range restriction (Bridger et al., 1998
; Hoshino et al., 1995
). In an attempt to identify the host range restriction determinants in rabbits, we recently tested 20 heterologous (non-lapine) viruses or reassortants for replication in rabbits (Ciarlet et al., 1998a
). Five additional heterologous rotavirus strains, human S2 (P1B[4], G2), avian Ty-1 (P[17], G7), avian Ch-2 (P[17], G7), bovine B223 (P8[11], G10) and porcine Gottfried (P2B[6], G4), and two reassortant rotavirus strains DxUK (P7[5], G1) and ST3xUK (P7[5], G4) did not replicate in rabbits (unpublished results). Among all 27 rotaviruses tested, only the simian RRV strain was efficient at replication in rabbits when compared to the other heterologous (non-lapine) rotavirus strains (Ciarlet et al., 1998a
). Although an inoculum dose of 2·4x108 p.f.u. of RRV results in a productive infection in rabbits (as measured by the magnitude and duration of virus antigen shedding and the magnitude of the immune response), the dose of RRV tested was 684 times greater than our standard challenge dose of lapine ALA (3·5x105 p.f.u.). Therefore, it remained to be determined if a lower dose of RRV would result in a productive infection in rabbits.
The replication kinetics of a particular rotavirus strain in a host species are likely to be determined by an overall combination of genes, making it difficult to dissect the individual gene(s) which control rotavirus host range restriction (Broome et al., 1993 ; Burke & Desselberger, 1996
; Ciarlet et al., 1998a
; Hoshino et al., 1995
). Simian rotavirus strains SA11 and RRV share similar VP7 (96%) and VP4 (88%), but the level of identity of NSP1 (57%) is low (Dunn et al., 1994a
). Unlike RRV, SA11 exhibits limited replication in rabbits at high doses (
108 p.f.u.) (Ciarlet et al., 1998a
). High levels of VP7 (9697%) and VP4 (6970 %) sequence identity exist between RRV and the lapine strains (Ciarlet et al., 1997
), but levels of identity with other gene products implicated in rotavirus host range restriction, particularly NSP1, have not been previously assessed because the corresponding gene sequences of the lapine rotavirus strains were not known. To determine the basis of the replication efficiency of RRV in rabbits, (i) rotavirus antibody-free rabbits were inoculated orally with 103, 105 or 107 p.f.u. of RRV to evaluate whether lower doses of RRV would productively infect rabbits and to obtain an estimate of the 50% infective dose (ID50), and (ii) the genes encoding the NSP1s of three lapine strains (ALA, C-11 and BAP-2) were sequenced and the predicted amino acid sequences were compared to all known NSP1 sequences.
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Methods |
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In vitro transcription, PCR, cloning and sequencing.
Single-stranded RNA transcripts were prepared from purified double-layered ALA, C-11 and BAP-2 particles as described (Ciarlet et al., 1997 ). The nucleotide sequences of gene 5 of ALA, C-11 and BAP-2 were determined by PCR which generated products of 1597 bp, corresponding to the full-length NSP1 of ALA and C-11, and a product of 1350 bp, corresponding to the NSP1 of BAP-2 lacking the 3' end of the gene. Briefly, reverse transcriptase was used to generate gene 5 complementary (c)DNA using a primer complementary to the 3' end of gene 5 of the simian rotavirus strain SA11 (5' GGGTTCACAGTATTTTGCCAGC 3'). Following purification of the first cDNA strand, PCR of gene 5 was achieved with the same 3' end primer and a primer complementary to the 5' end of gene 5 of the simian strain SA11 (5' GGGCTTTTTTTTGAAAAGTCTTG 3'). Amplified DNA was cloned into TA3pCR2.1 vector (Invitrogen). For accuracy in sequence determination, two independent clones from each rotavirus strain from individual PCR reactions were sequenced by the dideoxynucleotide chain or dye termination method. Additional primers were synthesized to complete the nucleotide sequencing of the genes. Their nucleotide positions on gene 5 of ALA, polarities (plus or minus sense) and sequences (5' to 3') were as follows: Max/150 (nt 509532, plus sense), CCAATCACTCTAAACGCTGCACTG; Max/330 (nt 10371060, minus sense), CTTACACTTGGAAATTGTCGAAGC; and Max/440 (nt 13551378, minus sense), AAGAGCTTCAGTTTTTAATATCAT. Confirmation of the DNA sequence was performed by sequencing both DNA strands of each of the different clones and by sequencing parts of the viral mRNA directly (Ciarlet et al., 1994
) using all three minus sense primers described herein. The 3' end sequences of the ALA and C-11 NSP1 genes are complementary to the PCR primer. The coding 3' end nucleotide sequence of the NSP1 gene of BAP-2 was determined from in vitro transcribed mRNA as described previously (Gorziglia et al., 1986
), using the primer complementary to the 3' end of gene 5 of the SA11 rotavirus strain. The non-coding 3' end nucleotide sequence of the NSP1 gene of BAP-2 was not determined.
Animals.
Three-month-old rotavirus- and specific-pathogen-free New Zealand White rabbits of either sex were obtained from Charles River Laboratories, Canada. To prevent complications due to Clostridium infections, 3 weeks prior to initiation of rotavirus studies, all rabbits were vaccinated once intramuscularly with a Clostridium spiroforme toxoid (kindly supplied by R. Carman, TechLabs, Blacksburg, VA) (C. ONeal, M. Ciarlet, R. J. Carman & M. E. Conner, unpublished results).
Animal inoculations and procedures.
Rabbits were inoculated orally with 2·4x103, 2·4x105 or 2·4x107 p.f.u. (corresponding to 4x102, 4x104 or 4x106 f.f.u., respectively) of simian RRV, with 1x107 or 3·5x105 p.f.u. (2x105 f.f.u.) of lapine ALA rotavirus, or with PBS as described (Ciarlet et al., 1998 a ; Conner et al., 1988
). All rabbits were individually housed and maintained in open cages for primary and challenge inoculations in a BL2 containment facility at Baylor College of Medicine. Rabbits inoculated with RRV or PBS were housed in a single room, while rabbits inoculated with ALA rotavirus were housed in a separate room. Experiments with ALA rotavirus and RRV were never performed concurrently. Cross-contamination of rabbits by different virus strains used in different experiments has never been encountered because each animal room and its entire contents are either heat- or chemically sterilized between experiments. Additionally, strict policies and procedures were followed for housing and handling the rabbits to prevent possible cross-contamination, and care and maintenance of the rabbits was performed by a single caretaker not exposed to other laboratory animals. Transmission efficiency of RRV in rabbits was also monitored by its ability to spread horizontally to mock-inoculated control animals housed in the same room and in the same rack, but in different cages from RRV-inoculated rabbits. In the previous study, transmission of RRV could not be assessed because the rabbits were housed in individual isolators under negative pressure (Ciarlet et al., 1998a
). All rabbits were challenged orally with 103 infectious doses (ID50) (3·5x105 p.f.u./ml) of lapine ALA rotavirus 28 days post-inoculation (p.i.) as described (Ciarlet et al., 1998a
; Conner et al., 1988
).
Collection of samples.
For detection of rotavirus-specific antibody responses, serum and faecal samples were collected at 0 and 28 days p.i. and 28 days post-challenge (p.c.) as described (Ciarlet et al., 1998a ; Conner et al., 1988
). Processing of faecal samples for antibody detection was performed as described (Ciarlet et al., 1998a
). To detect virus shedding, faecal samples were collected 010 days p.i. and 014 days p.c. and were processed as described (Ciarlet et al., 1998a
; Conner et al., 1988
).
Gastrointestinal transit time in rabbits.
To measure gastrointestinal transit time in rabbits, 3-month-old fasted rabbits (n=4) were inoculated orally with 10 ml of sterile H2O containing 30 small (1·5 mm) radiopaque markers using a blunt-ended feeding needle (Popper & Sons, New Hyde Park, NY). Faecal pellets were collected twice a day for 5 days, and transit time through the gastrointestinal tract was assessed by counting excreted markers at each time-point following radiography of the faecal material.
ELISA to measure rotavirus excretion and total antibody responses.
The ELISAs to measure rotavirus excretion and to measure total (IgA, IgM, IgG) antibody to rotavirus responses were performed as described (Ciarlet et al., 1998a ). A positive reaction was defined as an absorbance reading at 450 nm (A450)
0·1 for the antigen ELISA or a value
0·1 after subtracting A450 values of the antigen-negative well (mock) from the antigen-positive well for antibody ELISA (Conner et al., 1988
).
Analysis of rotavirus infection in rabbits.
Detection of virus antigen shedding after inoculation of heterologous viruses was defined as productive if faecal virus antigen shedding was comparable in magnitude and duration to homologous lapine ALA rotavirus infection, whereas limited replication was defined as when the amount and days of shedding was approximately two- to fourfold lower than the values obtained with homologous strains (productive infection) and antibody conversion occurred (Ciarlet et al., 1998a ). Minimal replication of a virus in rabbits was defined as antibody conversion after inoculation of the heterologous strain, but undetectable virus antigen shedding. Total protection from challenge was defined as no faecal shedding of virus antigen as detected by ELISA.
Analysis of RNA electropherotype by PAGE.
To confirm that rabbits shed only the virus with which they were inoculated, 10% faecal suspensions from virus-inoculated rabbits which were positive for rotavirus by ELISA or FFA were tested by PAGE. Nucleic acids of representative input and recovered virus from faecal material were extracted and subjected to electrophoresis in a 7% polyacrylamide gel and genome segments were visualized by silver staining (Ciarlet & Liprandi, 1994 ).
FFA to determine infectious virus titres and FFNA to measure neutralizing antibodies.
FFAs were performed as described (Ciarlet et al., 1998b ) on faecal samples collected following heterologous or homologous rotavirus inoculation of rabbits to compare infectious virus shedding relative to ELISA virus antigen shedding. Total infectious rotavirus shedding in faecal samples was calculated by the addition of individual titres of infectious virus in positive faecal samples and expressed in f.f.u./ml. An estimate of the net yield of virus output was calculated by multiplying the total infectious virus shed in faecal samples by the minimal volume (50 ml) of the entire (small, large and caecal) intestinal contents as determined previously (Conner et al., 1988
). Focus fluorescent neutralization assays (FFNAs) were performed as described (Ciarlet et al., 1994
), with the endpoint determined as the serum dilution producing a
66% reduction in the number of fluorescent foci.
Statistical analyses.
Statistical analyses of antibody titres between groups and correlation coefficients were performed by the MannWhitney U-test and by Pearsons correlation coefficient using SPSS version 7.5 for Windows (SPSS, Chicago, IL).
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Results |
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To compare infectious virus shedding relative to ELISA virus antigen shedding, faecal samples collected following ALA or RRV inoculation of a subset of rabbits were tested by FFA. The titres of homologous ALA at the peak of virus shedding ranged from 1·75x106 to 6x105 f.f.u./ml in rabbits inoculated with 3·5x105 p.f.u. (2x105 f.f.u.) of ALA (Fig. 2A). Peak titres of infectious heterologous RRV shed by the rabbits productively infected with either 2·4x107 or 2·4x105 p.f.u. (4x106 or 4x104 f.f.u., respectively) ranged from 5x105 to 4x105 f.f.u./ml, while those of the rabbits that exhibited limited infection ranged from 2x103 to 6x104 f.f.u./ml (Fig. 2B
, C
).
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To confirm that RRV replication, and not degradation or excretion of virus input, occurred in rabbits, the transit time through the rabbit gastrointestinal tract and an estimate of the net yield of virus output were determined. Radiopaque markers administered orally to 3-month-old fasted rabbits (n=4) were excreted in faeces between 10 and 26 h p.i. (data not shown). Therefore, since gastrointestinal transit time in rabbits is approximately 24 h and RRV was shed 46 days p.i., RRV antigen shedding must reflect virus replication, not degradation or excretion of virus input. The net yield of infectious virus was estimated knowing that the minimal volume of the entire (small, large and caecal) intestinal contents of age-matched ALA-infected rabbits is 50 ml (Conner et al., 1988 ). The net yield of ALA infectious virus in the rabbit intestine was at least 12 logs (1x1071·2x108 f.f.u.) over the inoculum (Table 1
). As with productively ALA-infected rabbits, RRV replication in productively RRV-infected rabbits exceeded input levels of RRV (Table 1
). The net virus yield shed in the faecal samples alone of the rabbit inoculated with 2·4x105 p.f.u. (4x104 f.f.u.) of RRV was 2·9x105 f.f.u./ml, surpassing the virus inoculum titre by more than 1 log, and the net virus yield would be 1·5x107 f.f.u., almost 3 logs in titre over input virus (Table 1
). Also, the net virus yield of the rabbit inoculated with 2·4x107 p.f.u. (4x106 f.f.u.) of RRV was estimated at 3·3x107 f.f.u. (1 log above virus input) (Table 1
). Hence, productive infection of rabbits by both ALA and RRV corresponds to net yield of virus titre over inoculum titre, indicating that replication of both viruses occurred. Limited replication corresponds to lower infectious virus titres compared to those obtained in a productive infection. In fact, net virus yield of rabbits that exhibited limited RRV replication either barely rose or did not rise above virus input titres, suggesting limited, and not productive, replication (Table 1
).
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To confirm that RRV was transmitted horizontally to the PBS-inoculated rabbit, we analysed the RNA electropherotype of the virus recovered in selected faecal samples of rabbits inoculated with ALA, RRV or PBS by PAGE. No experiments with lapine ALA rotavirus were concurrently performed with those of RRV or PBS. In faecal samples from separate experiments in which rabbits were inoculated with ALA or RRV, the recovered virus was always identical to the inoculum virus (Fig. 3 and data not shown). The RNA electropherotype of ALA is easily distinguishable from that of RRV since genome segment 11 of ALA is re-arranged (Fig. 3
). The RNA electropherotype observed in the faecal sample from the PBS-control rabbit that shed virus at 12 days p.i. was identical to that of RRV (Fig. 3
). Thus, RRV unequivocally spread to one of the control rabbits, confirming horizontal transmission of RRV in rabbits.
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Estimation of ID50 of RRV in rabbits
Based on virus antigen shedding or induction of an immune response, all eight rabbits inoculated with 2·4x107 (n=4) or 2·4x105 (n=4) p.f.u. of RRV were infected with RRV, whereas only one of three rabbits inoculated with 2·4x103 p.f.u. of RRV, became infected following inoculation. Based on these data, we estimated using the Karber equation that the ID50 of RRV in rabbits is 2·4x104 p.f.u. A more precise estimate of the ID50 of RRV in rabbits might be obtained if additional rabbits were inoculated with doses of 103 and 104 p.f.u. of RRV.
Protection induced by RRV against virus antigen shedding from challenge
Following challenge with the homologous ALA rotavirus strain, all the rabbits inoculated with ALA or with 105 and 107 p.f.u. of RRV were completely protected against infection based on lack of virus antigen shedding (data not shown) and lack of anamnestic responses (fourfold increases in titre) of either total or neutralizing antibody (Table 2
). The PBS-inoculated rabbit that was infected with RRV as a result of horizontal transmission of RRV was also 100% protected against challenge (Table 2
). The other two PBS-inoculated rabbits were not protected from challenge and each shed virus antigen for 5 days (data not shown) and consequently developed serum and intestinal rotavirus-specific antibody responses (Table 2
). None of the rabbits (n=4) inoculated with 103 p.f.u. of RRV were protected from challenge; all rabbits shed virus antigen, with a mean duration of 4·75 days (data not shown), and developed serum and intestinal rotavirus-specific antibody responses following challenge (Table 2
). As with previous results (Ciarlet et al., 1998a
), protection from ALA challenge correlated with the presence of a local immune response (P=0·001, r=0·765, Pearsons correlation coefficient) or a serological immune response (P<0·001, r=0·873, Pearsons correlation coefficient) (Table 2
). However, a low level systemic immune response in the absence of a local response was not enough to confer protection from challenge.
Sequencing of NSP1 from lapine rotaviruses
The fundamental structure of the NSP1 genes from the three lapine strains sequenced was similar to those of other rotavirus strains (Dunn et al., 1994a ; Fujiwara & Nakagomi, 1997
; Kojima et al., 1996
; Xu et al., 1994
). The gene of all three lapine NSP1s consisted of 1597 bp with one open reading frame beginning at nucleotide 32 and a single TAA stop codon at nucleotide 1515. The predicted size of the lapine NSP1 proteins was 492 amino acids, two amino acids less than the NSP1 of RRV (1601 bp, 494 aa) (Dunn et al., 1994a
). As with most rotavirus strains, the eight prolines (potentially important for conformation) and the cysteine-rich region (putative RNA binding domain) were conserved in all three rabbit strains.
A comparison of the complete deduced amino acid sequence for the genes encoding the NSP1 of the lapine strains with that of simian rotavirus strains is shown in Fig. 4. A high degree of amino acid identity (91·595·1%) was found among the NSP1 proteins of all three lapine rotavirus strains. The deduced amino acid sequences of the NSP1 genes of the lapine strains were also compared with the NSP1 sequences of representative rotavirus strains (Table 3
). The highest degree of identity was found with the simian RRV strain (85·387·5%) and with the feline FRV64 strain (8385%). With the remaining rotavirus strains examined, the amino acid identity with any lapine NSP1 ranged from 35·4% (bovine strain B223) to 71·6% (equine strains H-2, L338 and FI-23).
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Discussion |
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Studies by others have indicated that gene 5 sequences generally segregate according to the species of origin and represent the best association with species of origin and can best determine interspecies relatedness within the overall genomic RNA constellation (or genogroup) (Dunn et al., 1994a ; Fujiwara & Nakagomi, 1997
; Hua et al., 1994
; Kojima et al., 1996
; Nakagomi & Kaga, 1995
; Palombo & Bishop, 1994
). NSP1 has been implicated in rotavirus host range restriction in some studies (Broome et al., 1993
; Dunn et al., 1994a
; Fujiwara & Nakagomi, 1997
; Gombold & Ramig, 1986
; Kojima et al., 1996
), but not in others (Bridger et al., 1998
; Ciarlet et al., 1998a
; Gombold & Ramig, 1986
; Hoshino et al., 1995
). The role of NSP1 in rotavirus host range restriction may depend on the genetic background of a particular virus strain or the constellation of rotavirus genes, or alternatively, the host (Burke & Desselberger, 1996
; Ciarlet et al., 1998a
; Hoshino et al., 1995
). The genes encoding the NSP1s of three lapine rotavirus strains (ALA, C-11 and BAP-2) were sequenced and our results clearly indicate that there is a close relationship between the NSP1 of the lapine strains and the simian RRV strain (8588%). The amino acid identity of NSP1 of RRV and the lapine rotavirus strains is the highest reported yet for this protein from viruses from different species. Therefore, rabbits are possibly more susceptible to infection by RRV than by other heterologous rotavirus strains because of the similarity of the NSP1 sequences between RRV and the lapine rotaviruses. A close identity between the NSP1 of human and porcine strains has also been noted, albeit slightly lower (range 6986%) (Dunn et al., 1994a
; Palombo & Bishop, 1994
; Xu et al., 1994
). Recently, dot blot hybridization assays revealed that the NSP1s of canine (RS15, K9 and CU-1) and feline (FRV64 and Cat97, but not Cat2) rotaviruses share a high level of similarity (Fujiwara & Nakagomi, 1997
). In fact, the amino acid sequence of the NSP1 of FRV64 strain is very similar to that of RRV (85% amino acid identity) (Fujiwara & Nakagomi, 1997
). Therefore, we predict that FRV64 might be capable of replication in rabbits, but FRV64 was not available for testing.
The fact that the lapine strains and RRV share an unusually high amino acid identity (8588%) of gene 5, does not directly implicate gene 5 in host range restriction of rotaviruses. We showed previously that a reassortant rotavirus with ALA gene 5 and all other genes from simian SA11 replicated with less efficiency than either parental rotavirus strain (Ciarlet et al., 1998a ). In mice, gene 5 is implicated in rotavirus host range restriction (Broome et al., 1993
) and analysis of NSP1 amino acid sequences revealed that murine rotavirus strains (EW and EHP) are more closely related to RRV (5051%) than to other rotavirus strains (3740%) that do not replicate in mice (Broome et al., 1993
; Dunn et al., 1994a
; Kojima et al., 1996
). Since RRV and the murine strains and RRV and the lapine strains also share similar VP4s (7782% and 6970%, respectively) and VP7s (8890% and 9697%, respectively), which are both gene products associated with rotavirus host range restriction (Ciarlet et al., 1998a
; Dunn et al., 1994b
; Hoshino et al., 1995
), the overall similarity of their genes encoding NSP1, VP4 and VP7 may explain the replication competence of RRV in mice and in rabbits (Ciarlet et al., 1998a
). If our hypothesis is correct, then RRV may also replicate efficiently in cats and dogs due to the similarities of the NSP1s between RRV and canine and feline rotavirus strains, which also share the same VP7 serotype (G3), VP4 type (P[3]) as well as similar NSP4 type (Fujiwara & Nakagomi, 1997
; Horie et al., 1997
; Kirkwood & Palombo, 1997
; Ciarlet et al., 2000
).
Other genes not yet analysed for the lapine strains may also share high similarities with RRV, which might contribute to the replication efficiency of RRV in rabbits. Sequence comparison between RRV and lapine rotaviruses of the other rotavirus genes whose products have been implicated in rotavirus host range restriction (VP3, NSP2 and NSP4) cannot be performed because of lack of sequence availability; only the sequences of NSP2 and NSP4 of RRV have been reported. However, recent sequencing data of gene 10 (encoding NSP4) of lapine rotavirus strains indicate that the lapine NSP4s are closely related to each other, and they are divergent from that of RRV (Ciarlet et al., 2000 ). The low amino acid identity (5657%) shared between the NSP1 of SA11 rotavirus and the NSP1s of lapine rotavirus strains and RRV (Dunn et al., 1994a
) may explain why SA11 rotavirus, a strain that shares similar VP7 (9597%) and VP4 (6870 %) with the lapine rotavirus strains (Ciarlet et al., 1997
), exhibits limited replication in rabbits (Ciarlet et al., 1998a
).
Thus, whether susceptibility of rabbits to infection by RRV is due to NSP1 remains to be determined. Reassortants containing gene 5 from RRV and the other genes from a heterologous strain with limited replication in rabbits, and vice versa, could provide evidence for or against NSP1 as an important determinant for rotavirus host range in rabbits. It will be interesting to determine what unusual properties RRV possesses that allow it to replicate in a broad range of species (monkey, mouse, rabbit and human) and whether RRV will replicate efficiently in other species.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 August 1999;
accepted 20 January 2000.