Department of Virology, National Institute of Public Health, PO Box 4404 Torshov, N-0403 Oslo, Norway1
Ohio Agricultural Research and Development Center, Ohio, USA2
Moredun Research Institute, Edinburgh, UK3
Faculty of Medicine, University of Tokyo, Japan4
National Institute of Animal Health, Ibaraki, Japan5
Author for correspondence: Christine Jonassen. Fax +47 22 04 24 47. e-mail c.m.jonassen{at}folkehelsa.no
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human astroviruses (HAstV) are a common cause of diarrhoea in children, the elderly and the immunosuppressed (reviewed by Glass et al., 1996 ). They are non-enveloped particles with a plus-strand RNA genome of approximately 7 kb (Carter & Willcocks, 1996
). The genome contains three open reading frames (ORFs), as well as terminal non-coding regions (NCR). ORFs 1a and 1b are presumably linked by a translational frameshift, and encode non-structural proteins. ORF2 is transcribed into a subgenomic mRNA (Monroe et al., 1991
, 1993
; Cubitt, 1996
) and encodes the capsid proteins. HAstV is divided into different serotypes (Kurtz & Lee, 1984
) that are designated HAstV-1 to HAstV-8. The serotypes correspond well with phylogenetic reconstructions based on genome sequences (Belliot et al., 1997b
; Monceyron et al., 1997
).
Only limited sequence information has been available for animal astroviruses. A cat astrovirus (FAstV) ORF2 sequence is available in the nucleic acid databases, and recently full-length genomic sequences of an isolate of turkey astrovirus (TAstV-2) (Koci et al., 2000 ) and ANV (Imada et al., 2000
) were published.
The astrovirus capsid proteins are presumably synthesized as a single precursor (Lewis et al., 1994 ) that is subsequently processed, but the maturation process is not fully understood. Different experimental conditions, as well as strain-specific differences, may account for the differences in the capsid compositions reported. For HAstV-1 and -2 an initial capsid precursor of 8690 kDa is observed (Monroe et al., 1991
; Lewis et al., 1994
; Bass & Qiu, 2000
). This is processed to a 79 kDa protein, which, when cultured in the absence of trypsin, is the major HAstV-1 structural protein. However, the low infectivity of these particles suggests that further extracellular processing does occur in vivo, in the presence of trypsin, probably to three distinct proteins of 2034 kDa (Bass & Qiu, 2000
; Monroe et al., 1991
; Sanchez-Fauquier et al., 1994
). The N terminus of the 79 kDa HAstV-1 protein (Bass & Qiu, 2000
), as well as two HAstV-2 overlapping capsid proteins, VP26 and VP29 (Sanchez-Fauquier et al., 1994
), have been determined by amino acid sequencing. In other HAstV serotypes (Belliot et al., 1997a
; Kurtz & Lee, 1987
) and in OAstV (Herring et al., 1981
) and PAstV (Shimizu et al., 1990
) different numbers and sizes of capsid proteins are reported.
A 35 nt stemloop, previously referred to as s2m, near the astrovirus OFR2 stop codon, is a highly conserved RNA structure. Sequence differences in base-paired nucleotides of s2m are always accompanied by compensatory mutations that maintain the complementarity. The s2m is also present in the 3'-end of the genomes of avian infectious bronchitis virus and equine rhinovirus 2 (Jonassen et al., 1998 ). In the recently published ANV sequence, the s2m was also present (Imada et al., 2000
), but it was not present in TAstV-2 (Koci et al., 2000
).
The objective of the present work was to define common features of the astrovirus capsid proteins, and to reconstruct a phylogeny that could shed light on the dissemination and evolution of astroviruses.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequencing.
Isolation of RNA was performed either with Trizol (Gibco BRL) or QIAamp Viral RNA Mini kit (QIAGEN). Initially the 3'-ends of virus RNA were amplified and sequenced as described earlier (Jonassen et al., 1998 ). Longer amplicons were generated using the 5'-RACE kit (Gibco BRL) using a minus-strand primer based on the initially obtained 3'-sequence both for reverse transcription and in the subsequent 5'-RACE PCR. The PCR products obtained were sequenced in both directions using a primer walking strategy. Sequencing was performed using the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and analysed on an ABI PRISM 310 Genetic Analyser (Applied Biosystems).
Biocomputing.
Sequence analyses were performed using the Wisconsin sequence analysis package version 10.0 (Genetics Computer Group, Madison, Wisconsin, USA) and the PHYLIP package (Joseph Felsenstein, Department of Genetics, University of Washington, Seattle, Washington, USA; http://evolution.genetics.washington.edu/). These programs were accessed via the Norwegian EMBnet node (http://www2.no.embnet.org/). To avoid bias caused by over-representation of HAstV sequences, the weight of these sequences was reduced during aligning. Alignments were manually adjusted using the BioEdit sequence alignment editor (Tom Hall, Department of Microbiology, North Carolina State University, North Carolina, USA; http://www.mbio.ncsu.edu/RNaseP/home.html). Similarity plot analysis was done with the SimPlot program (Stuart C. Ray, Department of Medicine, Johns Hopkins University, Baltimore, Maryland, USA; http://www.med.jhu.edu/deptmed/sray/download/).
Accession numbers.
The sequences used in the figures have the following accession numbers: HAstV-1, S68561; HAstV-2, L06802; HAstV-3, AF117209; HAstV-4, Z33883; HAstV-5, HA15136; HAstV-6, Z46658; HAstV-7, Y08632; HAstV-8, Z66541; TAstV-1, Y15936; TAstV-2, AF206663; ANV, AB033998; OAstV, Y15937; PAstV, Y15938 and FAstV, AF056197.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The astrovirus ORF2 encodes between 671 aa in TAstV-1 and 816 aa in FAstV, corresponding to deduced molecular masses (Mr) of 72·5 kDa and 89·8 kDa respectively. ORF2 was shortest in the three avian astroviruses. The amino acid sequences inferred from the capsid genes of FAstV, PAstV, OAstV, TAstV-1, TAstV-2, ANV and the eight HAstV serotypes were aligned. An alignment of the N-terminal half of the putative capsid precursor of representative astroviruses is shown in Fig. 1. In the C-terminal half, the difference between the astroviruses was so extensive that the alignment is probably suboptimal and is therefore not shown. This is illustrated in Fig. 2
, in which the per cent identical residues along our best alignment are shown. As the C-terminal parts of the capsid precursor sequences were difficult to align, and the s2m is most likely homologous, the alignment was adjusted to keep the s2m aligned.
|
|
The 3'-NCR was about 80 nt in most of the astroviruses. In the avian astroviruses, however, it ranged from 140 nt in TAstV-1 to 305 nt in ANV (Imada et al., 2000 ). The ANV s2m was 117 nt farther from the poly(A) tail than the HAstV s2m. Imada et al. (2000)
calculated a 91% sequence identity between the ANV s2m and other astrovirus s2m, but all the differences were in base-paired nucleotides which covaried to maintain the RNA folding predicted earlier (Monroe et al., 1993
; Jonassen et al., 1998
). Except for the avian astroviruses, the ORF2 stop codon was located in the middle of the s2m. In TAstV-1 and ANV the stop codons were respectively 21 and 103 nt upstream of this position.
The basic residues in the N-terminal part of the capsid precursor, described as being similar to basic stretches found in the capsid proteins of two coronaviruses (Carter, 1994 ), were highly conserved between HAstV, FAstV, PAstV and TAstV-2 (position 1934 in Fig. 1
). A pattern of alternating small and basic amino acids, mainly serine (S) and arginine (R), was apparent in these sequences, most significantly in TAstV-2, where an SR dipeptide was repeated six times. In OAstV, ANV and TAstV-1 the homologous residues were also highly basic, but otherwise different from the astrovirus consensus sequence.
The N terminus of the 79 kDa capsid protein found in HAstV-1 cultivated without trypsin (Bass & Qiu, 2000 ) is at position 75 in Fig. 1
. In this region the amino acid sequences of HAstV, FAstV and PAstV were highly conserved, while the avian astroviruses and OAstV diverged from the consensus. The N termini of the HAstV-2 structural proteins VP29 and VP26 reported by Sanchez-Fauquier et al. (1994)
are in positions 379 and 416, respectively, in Fig. 1
. These putative trypsin cleavage sites do not seem to be conserved in all astroviruses.
A similarity plot comparing the amino acid sequence of HAstV-2 with HAstV-7 and the animal astroviruses is shown in Fig. 2. ANV and TAstV-2 showed profiles similar to TAstV-1, but were not included in the figure. The N-terminal half of the capsid precursor was considerably more conserved than the C-terminal half. In the N-terminal half and the C-terminal end of the capsid precursor, FAstV was as close to HAstV-2 as HAstV-7 was. The same profile of similarity with HAstV-2 was seen for PAstV, while OAstV did not display the C-terminal similarity. As is also shown in Table 1
, the overall similarities to the HAstV sequences decreased in the following order: FAstV>PAstV >OAstV>avian astroviruses.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Viruses infecting different hosts have either coevolved with the hosts since the hosts diverged, or they are the result of inter-species transmission. The phylogenetic tree (Fig. 3) reveals that HAstV, FAstV and PAstV are more closely related to each other than to OAstV. Sheep, cats and pigs all parted with human ancestors some 100 million years ago. As the latest common ancestor of sheep and pigs presumably is more recent (Novacek, 1992
), the most likely explanation for the discrepancy between the family trees of the viruses and their hosts is that astroviruses have been transmitted between cats, pigs and humans, possibly involving intermediary hosts. The observation that FAstV is as related to HAstV as the most distantly related HAstVs are to each other suggests that zoonoses involving pigs, cats and humans have occurred relatively recently compared to the latest common ancestor of all astroviruses. It follows that although zoonoses appear to be rare, one should be aware of the possibility and take proper precautions.
The absence of the s2m from TAstV-2 may reflect that the TAstV-2 lineage split from the other astroviruses before an ancestor of the other astroviruses acquired the s2m. Alternatively, the s2m was once present in TAstV-2 as well, but was later lost. The present data do not support one of these explanations over the other.
It has proven feasible to detect all HAstV serotypes with a single PCR (Jonassen et al., 1995 ; Sakon et al., 2000
). Conserved nucleotide elements could facilitate the design of an Astroviridae-specific PCR to be used for the detection of viruses from animals for which no sequence information is available. Alignment of the nucleotide sequences from the 3'-end of the available astrovirus genomes revealed that the only viable option for a pan-astrovirus PCR rests with the s2m, in combination with a downstream primer that binds to the poly(A) tail. A PCR with an s2m sense primer and an NV(T)n anti-sense primer may function, even for Astroviridae other than the ones sequenced. One should keep in mind, however, that the s2m is present in certain other viruses (Jonassen et al., 1998
), while it is absent in TAstV-2.
As most astroviruses share the same morphology, we expected to find conserved features in the capsid proteins. Conversely, the presence of motifs conserved at the amino acid level suggests an essential function, and thus possibly a role in the assembly and function of the virus particle.
It has been suggested that in the case of HAstV, unlike Norwalk virus, post-translational processing is required for the assembly of virus particles (Carter & Willcocks, 1996 ). In HAstV-2 cultured in the presence of trypsin, the three structural proteins are referred to as VP34, VP29 and VP26, where VP26 is an N-terminal truncated version of VP29 (Sanchez-Fauquier et al., 1994
). Bass & Upadhyayula (1997)
found two neutralizing monoclonal antibodies that reacted only with VP29, while a third also attached to VP26. Another neutralizing antibody developed by Sanchez-Fauquier et al. (1994)
reacted with both proteins. The results suggest that VP26 and VP29 contain at least one common motif on the surface of the virus capsid that may be involved in cell binding, while the N-terminal part of VP29 has an additional epitope of similar characteristics. The lack of cross-reactivity with monoclonal antibodies against VP26/VP29 suggests that the third, larger structural protein (VP34) is independently coded. The HAstV peptide between the suggested N terminus of the 79 kDa protein (Bass & Qiu, 2000
) and the N terminus of the 29 kDa protein (Sanchez-Fauquier et al., 1994
) has a deduced Mr of 31 kDa, which may be compatible with VP34. The peptide from the N terminus of the 29 kDa protein to the end of ORF2 has a deduced Mr of 4649 kDa, which probably does not give room for a 34 kDa protein between the C terminus of VP29 and the end of ORF2. The observation that the neutralizing monoclonal antibodies all react to VP26/VP29, rather than to VP34, suggests that VP26/VP29 dominates the surface of the particle. This may explain the higher heterogeneity of VP26/VP29 compared to the N-terminal part of the capsid precursor.
Unlike other astroviruses, ANV can be cultured to high titres in the absence of trypsin. This suggests that the processing of the capsid precursor is different from that of other astroviruses, and may explain the lack of conservation of the trypsin cleavage sites that are suggested for HAstV. The lack of requirement for trypsin-like proteases may be related to the different tissue tropism of ANV compared to most astroviruses.
The basic region in the N-terminal part of the capsid precursor of the astroviruses contains, to a variable extent, an SR-repeat motif. In addition to certain coronaviruses that are related to transmissible gastroenteritis virus, SR repeats are also found in, for example, the E2 protein of some human papillomaviruses (Lai et al., 1999 ) and some baculoviruses (Oellig et al., 1987
). In multicellular organisms long stretches of alternating S and R residues are found in the SR protein family of pre-mRNA splicing factors. The SR region of the papillomavirus E2 protein has been shown to be important for nuclear localization and regulation of gene expression. It is possible that this motif serves a regulatory function also in the astroviruses.
The region beyond the presumptive 3'-end of the VP26/VP29 gene is hypervariable, with large inserts or deletions. The C-terminal 6 aa are highly conserved, with the exception of the avian astroviruses. This motif, however, is within the s2m, and is thus presumably conserved by selection at the RNA level. This assumption is strengthened by the location of the s2m downstream of ORF2 in TAstV-1 and ANV.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bass, D. M. & Qiu, S. (2000). Proteolytic processing of the astrovirus capsid. Journal of Virology 74, 1810-1814.
Belliot, G., Laveran, H. & Monroe, S. S. (1997a). Capsid protein composition of reference strains and wild isolates of human astroviruses. Virus Research 49, 49-57.[Medline]
Belliot, G., Laveran, H. & Monroe, S. S. (1997b). Detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region. Archives of Virology 142, 1323-1334.[Medline]
Carter, M. J. (1994). Genomic organization and expression of astroviruses and caliciviruses. Archives of Virology Suppl. 9, 429-439.[Medline]
Carter, M. J. & Willcocks, M. M. (1996). The molecular biology of astroviruses. Archives of Virology Suppl. 12, 277-285.[Medline]
Cubitt, W. D. (1996). Historical background and classification of caliciviruses and astroviruses. Archives of Virology Suppl. 12, 225-235.[Medline]
Glass, R. I., Noel, J., Mitchell, D., Herrmann, J. E., Blacklow, N. R., Pickering, L. K., Dennehy, P., Ruiz-Palacios, G., de Guerrero, M. L. & Monroe, S. S. (1996). The changing epidemiology of astrovirus-associated gastroenteritis: a review. Archives of Virology Suppl. 12, 287-300.[Medline]
Gough, R. E., Collins, M. S., Borland, E. & Keymer, L. F. (1984). Astrovirus-like particles associated with hepatitis in ducklings. Veterinary Record 114, 279.
Herring, A. J., Gray, E. W. & Snodgrass, D. R. (1981). Purification and characterization of ovine astrovirus. Journal of General Virology 53, 47-55.[Abstract]
Hoshino, Y., Zimmer, J. F., Moise, N. S. & Scott, F. W. (1981). Detection of astroviruses in feces of a cat with diarrhea. Brief report. Archives of Virology 70, 373-376.[Medline]
Imada, T., Yamaguchi, S., Mase, M., Tsukamoto, K., Kubo, M. & Morooka, A. (2000). Avian nephritis virus (ANV) as a new member of the family Astroviridae and construction of infectious ANV cDNA. Journal of Virology 74, 8487-8493.
Jiang, B., Monroe, S. S., Koonin, E. V., Stine, S. E. & Glass, R. I. (1993). RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis. Proceedings of the National Academy of Sciences, USA 90, 10539-10543.[Abstract]
Jonassen, T. Ø., Monceyron, C., Lee, T. W., Kurtz, J. B. & Grinde, B. (1995). Detection of all serotypes of human astrovirus by the polymerase chain reaction. Journal of Virological Methods 52, 327-334.[Medline]
Jonassen, C. M., Jonassen, T. Ø. & Grinde, B. (1998). A common RNA motif in the 3' end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus. Journal of General Virology 79, 715-718.[Abstract]
Kjeldsberg, E. & Hem, A. (1985). Detection of astroviruses in gut contents of nude and normal mice. Brief report. Archives of Virology 84, 135-140.[Medline]
Koci, M. D., Seal, B. S. & Schultz-Cherry, S. (2000). Molecular characterization of an avian astrovirus. Journal of Virology 74, 6173-6177.
Kozak, M. (1997). Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO Journal 16, 2482-2492.
Kurtz, J. B. & Lee, T. W. (1984). Human astrovirus serotypes. Lancet ii, 1405.
Kurtz, J. B. & Lee, T. W. (1987). Astroviruses: human and animal. CIBA Foundation Symposia 128, 92-107.[Medline]
Lai, M. C., Teh, B. H. & Tarn, W. Y. (1999). A human papillomavirus E2 transcriptional activator. The interactions with cellular splicing factors and potential function in pre-mRNA processing. Journal of Biological Chemistry 274, 11832-11841.
Lee, T. W. & Kurtz, J. B. (1994). Prevalence of human astrovirus serotypes in the Oxford region 197692, with evidence for two new serotypes. Epidemiology and Infection 112, 187-193.[Medline]
Lewis, T. L., Greenberg, H. B., Herrmann, J. E., Smith, L. S. & Matsui, S. M. (1994). Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein. Journal of Virology 68, 77-83.[Abstract]
McNulty, M. S., Curran, W. L. & McFerran, J. B. (1980). Detection of astroviruses in turkey faeces by direct electron microscopy. Veterinary Record 106, 561.[Medline]
Madeley, C. R. & Cosgrove, B. P. (1975). 28 nm particles in faeces in infantile gastroenteritis. Lancet ii, 451452.
Monceyron, C., Grinde, B. & Jonassen, T. Ø. (1997). Molecular characterisation of the 3'-end of the astrovirus genome. Archives of Virology 142, 699-706.[Medline]
Monroe, S. S., Stine, S. E., Gorelkin, L., Herrmann, J. E., Blacklow, N. R. & Glass, R. I. (1991). Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells. Journal of Virology 65, 641-648.[Medline]
Monroe, S. S., Jiang, B., Stine, S. E., Koopmans, M. & Glass, R. I. (1993). Subgenomic RNA sequence of human astrovirus supports classification of Astroviridae as a new family of RNA viruses. Journal of Virology 67, 3611-3614.[Abstract]
Novacek, M. J. (1992). Mammalian phylogeny: shaking the tree. Nature 356, 121-125.[Medline]
Oellig, C., Happ, B., Muller, T. & Doerfler, W. (1987). Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81·2 to 85·0) of the Autographa californica nuclear polyhedrosis virus genome. Journal of Virology 61, 3048-3057.[Medline]
Reynolds, D. L. & Saif, Y. M. (1986). Astrovirus: a cause of an enteric disease in turkey poults. Avian Diseases 30, 728-735.[Medline]
Sakon, N., Yamazaki, K., Utagawa, E., Okuno, Y. & Oishi, I. (2000). Genomic characterization of human astrovirus type 6 Katano virus and the establishment of a rapid and effective reverse transcriptionpolymerase chain reaction to detect all serotypes of human astrovirus. Journal of Medical Virology 61, 125-131.[Medline]
Sanchez-Fauquier, A., Carrascosa, A. L., Carrascosa, J. L., Otero, A., Glass, R. I., Lopez, J. A., San Martin, C. & Melero, J. A. (1994). Characterization of a human astrovirus serotype 2 structural protein (VP26) that contains an epitope involved in virus neutralization. Virology 201, 312-320.[Medline]
Shimizu, M., Shirai, J., Narita, M. & Yamane, T. (1990). Cytopathic astrovirus isolated from porcine acute gastroenteritis in an established cell line derived from porcine embryonic kidney. Journal of Clinical Microbiology 28, 201-206.[Medline]
Snodgrass, D. R. & Gray, E. W. (1977). Detection and transmission of 30 nm virus particles (astroviruses) in faeces of lambs with diarrhoea. Archives of Virology 50, 287-291.
Tzipori, S., Menzies, J. D. & Gray, E. W. (1981). Detection of astrovirus in the faeces of red deer. Veterinary Record 108, 286.[Medline]
Williams, F. P.Jr (1980). Astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups. Archives of Virology 66, 215-226.[Medline]
Woode, G. N. & Bridger, J. C. (1978). Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves. Journal of Medical Microbiology 11, 441-452.[Abstract]
Received 8 September 2000;
accepted 12 January 2001.