Departamento de Genética y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Colonia Miraval, Cuernavaca, Morelos 62250, Mexico1
Author for correspondence: Ernesto Méndez. Fax +52 73 17 2388. e-mail ernesto{at}ibt.unam.mx
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Based on the virus reactivity with polyclonal antibodies, human astroviruses (HAstVs) have been classified into seven serotypes (HAstV-1 to -7) (Herrmann et al., 1988 ). Phylogenetic grouping of HAstV based on nucleotide sequence analysis of a limited region of ORF2 (Belliot et al., 1997
; Noel et al., 1995
) showed a good correlation between genotype and serotype, and recently the existence of an eighth HAstV serotype has been suggested based on genotyping of an HAstV strain isolated in UK (Belliot et al., 1997
). Only three additional strains of this genotype 8 astrovirus, isolated in Australia, Uganda and Ghaza, have been reported, which is why it is considered to be a rare serotype (Mustafa et al., 2000
). In a recent multicentric study carried out in Mexico (unpublished), it was found that six HAstV serotypes (1 to 4, 6 and 8) co-circulated in a single period of 6 months, with serotype 1 being the most frequent, as found in other studies from different geographical regions (Gaggero et al., 1998
; Noel & Cubitt, 1994
; Palombo & Bishop, 1996
; Shastri et al., 1998
). In one of the locations (Mérida City, Yucatan) included in the Mexican study, three of the eight HAstV strains detected belonged to genotype 8 (assignment made based on the sequence of the 3'-end of ORF2), suggesting that these strains might be more epidemiologically relevant than previously recognized. In this work, we adapted one of the genotype 8 HAstV strains (named Yuc-8) to grow in CaCo-2 cells, and determined its complete genomic nucleotide sequence, the first for an HAstV-8 strain.
For isolation of the virus, a stool sample was diluted 1:2 in PBS, extracted with Freon, and filtered through a 0·22 µm membrane. This material was treated with trypsin (10 µg/ml) for 1 h at 37 °C, and inoculated into a CaCo-2 cell monolayer. After 60 min, the inoculum was removed, fresh Eagles minimum essential medium was added, and the cells were incubated at 37 °C for 3 days. The virus was harvested by three freezethaw cycles, and was passaged again in the same cells, as described above. After seven passages, the presence of the virus was confirmed by immune electron microscopy with a hyperimmune serum to HAstV-1 (Herrmann et al., 1990 ); by ELISA (IDEIA Astrovirus, Dako); by immunocytochemistry with monoclonal antibody 8G4, which recognizes HAstV-1 to -7 (Bass & Upadhyayula, 1997
); and by RTPCR with oligonucleotides Mon244 and Mon245 (Noel et al., 1995
). Total RNA from Yuc-8-infected cells was obtained with Trizol (Gibco-BRL), and used as template to amplify the astrovirus genome by RTPCR. Reverse transcriptase SuperScript II (Gibco-BRL) and Vent DNA polymerase (New England Biolabs) were used for the RTPCR reactions. Oligonucleotides synthesized were initially based on the previously reported sequence for HAstV-1 (accession no. Z25771), and subsequently based on the sequence obtained from Yuc-8. The amplified DNA fragments were sequenced with an ABI Prism DNA automatic sequencer, model 377-18 (Perkin Elmer). The sequence of the 5' non-translated region (NTR) was determined from a PCR fragment obtained with an upstream 20-mer oligonucleotide corresponding to the 20 5'-end nucleotides which are conserved among astrovirus serotypes 1, 2 and 3. Nucleotide sequence of the 3' NTR was determined from a DNA fragment obtained by RTPCR with oligo(dT) and oligonucleotide Beg (Saito et al., 1995
) as primers. At least three PCR fragments of a given region, amplified independently, were used for determining the consensus sequence.
The full-length genomic RNA of HAstV Yuc-8 consists of 6759 bases, followed by a poly(A) tract. It has a 5' NTR and a 3' NTR of 83 and 85 nucleotides, respectively, and it is organized in three sequential open reading frames corresponding to ORFs 1a, 1b and 2 (Jiang et al., 1993 ; Willcocks et al., 1994b
). The encoded polyproteins have the characteristic motifs described above.
Comparison of the non-structural polyprotein region of astrovirus Yuc-8 with the corresponding sequence of viruses belonging to serotypes 1, 2 and 3 showed a high level of conservation among these strains (higher than 93% identity at amino acid level). A small region of high variability was found around amino acids 767 and 790 in ORF1a (numbering according to the Yuc-8 sequence), where 7 and 15 residues were missing in Yuc-8 and HAstV-2, as compared to HAstV-3 and HAstV-1, respectively (Fig. 1a, regions in/del I and in/del II, respectively). The absence of the 15 amino acid residues at position 790 (region in/del II) has been associated with the adaptation of astroviruses to HEK and LLCMK2 cells, since viruses grown in these cells, but not in CaCo-2 cells, lack that region (Willcocks et al., 1994a
). Yuc-8 is also missing those 15 residues despite its having been adapted to grow in CaCo-2 cells, which suggests that the in/del region II, if it plays a role, is not the only factor involved in adaptation of human astroviruses to a specific cell line. A more detailed analysis of field and culture-adapted astrovirus strains is needed to resolve this issue.
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Alignment of the ORF2 sequences of seven HAstV serotypes (Fig. 1b) showed the existence of two distinct domains, which were first observed by Willcocks et al. (1995)
who analysed three serotypes only. One conserved amino-terminal domain spans amino acid residues 1 to 415, with identities higher than 81% among the different serotypes. Two short variable regions between residues 292 to 319 (VR1) and 386 to 399 (VR2), not reported previously, were observed in this domain (Fig. 1b
). The highly variable second domain (identities of 36 to 60% among serotypes) starts at amino acid 416 and extends to the end of the protein, but in contrast to the report of Willcocks et al. (1995)
, the conservation at the end of the ORF2 was not observed among the serotypes (Fig. 1b
).
Phylogenetic analysis of ORF2 showed a completely different genetic relatedness among serotypes 1 to 8 when either of the two domains described above was analysed. The phylogenetic tree generated by comparison of the conserved domain (Fig. 2a) was very similar to the trees reported previously by Belliot et al. (1997)
and Noel et al. (1995)
, who analysed a region of 137 amino acids (residues 73 to 210 of Yuc-8). Serotypes 2, 4 and 8 were found to be closely grouped, while serotypes 1, 3, 5 and 6 were more distantly related to this group (Fig. 2a
). On the other hand, the dendrogram generated by comparison of the variable domain of ORF2 (residues 416 to 782) was quite different. Serotypes 4 and 8 were among the least related (Fig. 2b
), which resulted in a phylogenetic tree similar to that reported in another analysis including part of the 3'-end of the astrovirus genome (Monceyron et al., 1997
). It is likely that the variable carboxy-terminal domain of the ORF2 polyprotein is subjected to immunological pressure, which probably contributes to the variability observed. In fact, neutralizing antibodies which recognize VP29 and VP26 have been identified (Bass & Upadhyayula, 1997
). Furthermore, it is of interest that the genetic relatedness observed among the various HAstV serotypes, when the variable region was analysed, differs so much from the relationships found in the conserved amino-terminal half of the protein. The selective pressure that created these marked deviations from the mutation rate of astroviruses could operate differentially along the ORF2 polyprotein, most likely as the result of a sum of factors including, among others, the intrinsic structural constraints of the virus particle and the host immune response of the particular populations infected. On the other hand, the non-superimposable dendrograms shown in Fig. 2
could also be the result of intraserotypic astrovirus recombination, suggested as previously (Belliot et al., 1997
; Jonassen et al., 1998
). Whatever the reason for the observed variations, it is evident that the genomic region chosen to compare different HAstV strains should take into account the differential variability observed in the astrovirus genome, particularly that of ORF2.
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References |
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Received 9 May 2000;
accepted 8 August 2000.