INTRODUCTION

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Avian nephritis virus (ANV) acts as an etiological agent of growth retardation of young chickens by causing interstitial nephritis (15). The virus was first isolated from young chickens with chicken kidney (CK) cells in 1976 by our group (52). ANV is widely distributed in chickens worldwide, and turkeys may also be susceptible (5, 16). Field viruses of ANV exhibit different degrees of pathogenicity in chickens, producing results ranging from subclinical infection to death, and there are at least two serotypes (7, 14, 43).

ANV is an unclassified small round nonenveloped virus 28 nm in diameter and stable at pH 3.0; however, the buoyant density has not been determined because ANV is fragile in cesium chloride (CsCl). Growth inhibition of ANV by adding the nucleoside analog 5-bromo-2-deoxyuridine indicated that it has an RNA genome (52). The virus can replicate in the kidneys and intestine in vivo. Similar to picornaviruses, ANV does not require trypsin for in vitro cultivation. Based on pathological and immunologic data, however, ANV is distinct from avian encephalomyelitis virus and duck hepatitis viruses, which are members of avian enterovirus-like viruses of the family Picornaviridae (5, 28, 30, 31). These data led investigators to believe that ANV may belong to the genus Enterovirus of the family Picornaviridae, although neither the nucleotide nor the amino acid sequences had been reported yet (14). Small rounded nonenveloped RNA viruses (approximately 30 nm in diameter) are causative agents of gastroenteritis in animals and humans (23). Not only picornaviruses but also astroviruses are included as the agents (31, 33, 35, 53).

Astroviruses were first identified in diarrheal stools of children with gastroenteritis by electron microscopy (27). Astroviruses are small nonenveloped RNA viruses, approximately 28 nm in diameter, characterized by a star-like morphology (26). Astroviruses require trypsin to grow in cell culture (3, 42, 47, 50), which may have some implications for gastroenteritis. There are at least eight serotypes of human astroviruses (HAst). The genome consists of a positive-strand RNA of approximately 6,800 nucleotides (nt) with three open reading frames (ORFs). The first ORF (ORF 1a), encodes a 3C-like serine protease motif, and the second ORF (ORF 1b) encodes an RNA-dependent RNA polymerase (RdRp) motif; however, the astrovirus lacks a helicase motif typical of other positive-strand RNA viruses. ORF 1a is linked to ORF 1b by a ribosomal frameshifting motif, and both ORFs are probably translated from the genonic RNA. The third ORF (ORF 2) encodes a precursor polyprotein which is proteolytically processed to viral structural proteins. ORF 2 is likely expressed from a subgenonic RNA which is coterminal with the genonic RNA at the 3' end (3, 33, 34).

Definitive classification of ANV requires genetic information. In this study, the complete RNA genome was molecularly cloned and sequenced. Sequence analysis data indicated that ANV is a new member of the family Astroviridae. ANV is the first avian astrovirus whose genome has been completely sequenced. The infectious cDNA clone developed here is a tool to identify the critical regions for pathology and serology on the ANV genome.

	MATERIALS AND METHODS

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Cells and virus. Primary CK cell cultures were prepared from specific-pathogen-free (SPF) chickens (line PDL-1) (8) and were maintained in Eagle's minimal essential medium (EMEM) supplemented with antibiotics and 5% newborn bovine serum (15). Baby hamster kidney (BHK) cells were grown in EMEM containing 10% fetal calf serum. ANV (G-4260 strain) was plaque-purified through three consecutive cycles before use. Chicken antiserum to the G-4260 strain obtained by immunization of SPF chicks with the plaque-purified virus was used for fluorescent antibody (FA) tests (15).

EM observation. Viral particles purified from the supernatants of infected CK cells and an ultrathin section of infected cells were observed with an electron microscope (EM; JEM-100CX; Nippon Denshi Co., Tokyo, Japan). Infected cell culture fluid was harvested when the cells exhibited a maximum cytopathic effect (CPE) at 36 to 48 h postinoculation and were concentrated with polyethylene glycol 6000 (9). After centrifugation (8,000 × g for 30 min), the pellet was suspended in phosphate-buffered saline and loaded onto a 10 to 45% continuous linear sucrose gradient prepared in a polyalomer SW40 ultracentrifuge tube. The sample was ultracentrifuged on the sucrose gradient for 3.5 h at 36,000 rpm (SW-40Ti; Beckman) at 4°C; settings for acceleration and decelation were both "7." The fraction with the highest infectivity was obtained and examined for virus with an EM, after it was stained with 3% uranyl acetate (pH 7.0). For the preparation of ultrathin sections, infected CK cells were collected before the maximum CPE appeared (20 h p.i.), fixed with 2% glutaraldehyde in phosphate buffer, postfixed with 1% osmium tetroxide in phosphate buffer, dehydrated in a graded ethanol series, and embedded in an Epon mixture. Ultrathin sections were cut with a glass knife, stained with a lead citrate-uranyl acetate solution, and observed with an EM.

Isolation of ANV RNA. To clone the ANV genome, the culture fluid of ANV-infected cells was purified by sucrose gradient ultracentrifugation because several attempts to clone the virus from cesium chloride gradient-purified viral particles were unsuccessful. The viral antigen was monitored at each step with the EM. The virion RNA was extracted by using ISOGEN-LS total RNA isolation reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's recommendation.

Cloning the genome of ANV and sequencing. Standard molecular biology procedures were used to clone ANV cDNAs (41). Briefly, cDNA synthesis was performed using a Time Saver cDNA synthesis kit (Amersham Pharmacia Biotech), the reverse transcription of ANV RNA being primed by oligo(dT)_12-18 or by random six-residue primers. Second-strand synthesis produced double-stranded cDNA, which was subsequently ligated into the EcoRI site of the pBluescript II (pBSII) KS+ vector (Stratagene, La Jolla, Calif.). This was used to transform the Escherichia coli DH5 strain. The remainder of the genonic cDNA was cloned by three successive rounds of primer extension using synthetic primers derived from the sequence at the termini of the preceding clones. Clones containing the 5' end of the genome were obtained with the 5'-Full rapid amplification of cDNA ends (RACE) core kit (Takara, Tokyo, Japan). The cycle sequencing reaction was performed with a dye terminator cycle sequencing FS Ready Reaction kit (Perkin-Elmer Cetus) using M13 forward, M13 reverse, and custom primers and was analyzed with an ABI 373 automatic sequencer. The sequences of all clones derived from each round of primer extension and 5'-Full RACE were determined for both strands of the cDNA.

RNA blot hybridization. Two probes, one the 5'-end side (7FB-7RB; 302 bp at ORF 1a) and the other the 3'-end side (1F-1R; 280 bp at ORF 2 and the 3'-end nontranslated region [NTR]) (Table 1; Fig. 3), were synthesized with PCR DIG probe synthesis kits (Boehringer Mannheim). Total cytoplasmic RNA isolated from ANV-infected and uninfected cells at 16 and 24 h p.i. was resolved in a 1.0% agarose gel containing 0.1% sodium dodecyl sulfate, transferred to a nylon membrane, and probed with digoxigenin (DIG)-labeled DNAs. DIG nucleic acid detection kits (Boehringer Mannheim) were used according to the manufacturer's recommendations.

Comparative sequence analysis. Both nucleotide and deduced amino acid sequences were compared, the multiple alignments were done, the phylogenetic trees of ORF 1b and ORF 2 were constructed, and the potential secondary structures of the ANV genome were examined using the software of GENETYX-WIN, version 4 (Software Development Co., Ltd., Tokyo, Japan). The following nucleotide sequences were obtained from GenBank/EMBL/DDBJ: HAst serotype 1 (HAst 1), Z25771 and L23513; HAst 2, L13745; feline astrovirus, AF056197; porcine astrovirus, AB037272; turkey astrovirus, AF206663.

Construction of full-length cDNA clone for ANV. A full-length cDNA copy of ANV (G-4260 strain) was assembled in pBSII by sequentially linking overlapping fragments of the ANV cDNA at unique restriction sites (Fig. 1). These overlapping cDNA fragments were selected from the clones generated by cloning the genome of ANV for sequencing. Table 1 lists the PCR primers used for cloning clone 615 and the 5'-most primer on the viral genome comprising a T7 promoter as well as a BamHI site. The pBSII clone containing the full-length ANV cDNA between the BamHI and the NotI sites was named pANV-750. This plasmid harbors two T7 promoters organized in the same direction, one derived from the pBSII vector and the other derived from the 5'-most PCR primer. In the process of construction of pANV-750 from clone 750 and the PCR product (Fig. 1), a point mutation (A to G at nt 281; amino acid change Thr to Ala) was introduced by PCR. Finally, the vector plasmid was changed to pCMV vector (Clontech Laboratories, Inc., Palo Alto, Calif.) from pBSII at the NotI site and named pCMV-750.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Construction of the full-length ANV cDNA clone and infectivity. Open boxes, partial and full-length viral cDNA; solid box, RNA transcribed in vitro from pANV-750; arrowheads, T7 promoter; CMV, human cytomegalovirus immediate-early gene promoter; star, point mutation introduced by PCR; small arrow, primer direction and position (see Table 1); , negative for infectious virus; +, positive for infectious virus (see Fig. 7). In vitro transcripts prepared from the full-length ANV cDNA clone or a plasmid which can transcribe the genonic-size ANV RNA under the control of the CMV promoter were transfected onto CK or BHK cells using Lipofectin reagent (Bethesda Research Laboratories). The cells were tested for the presence of ANV antigens by FA tests 2 days after transfection.

Demonstrating infectivity of RNA transcribed from the cDNA clone (pANV-750) and cDNA of ANV. The ANV cDNA was positioned downstream of a viral T7 DNA-dependent RNA polymerase promoter, and the NotI restriction site allowed the linearization of the DNA immediately downstream of the viral poly(A) sequence. In vitro transcription with T7 polymerase (Promega, Madison, Wis.) was performed according to the manufacturer's recommendations in the presence of an m7G(5')ppp(5')G RNA cap structure analog and was followed by treatment with an RNase-free DNase (10).

Nucleotide sequence accession number. The complete nucleotide sequence of ANV has been submitted to the DDBJ, EMBL, and GenBank databases under accession no. AB033998.

	RESULTS

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Sequence analysis. The genonic RNA of ANV was 6,927 nt in length, excluding the poly(A) tail. The genome possesses three overlapping ORFs, ORF 1a, 1b, and 2 (Fig. 2; Table 2). When the virus uses the first methionine, ORF 1a (nt 14 to 3028) may encode a polypeptide of 1,005 amino acids (aa). ORF 1b (nt 3019 to 4548) may encode a polypeptide of 483 aa; it overlaps ORF 1a by 10 nt and was in reading frame +1, and its first AUG codon was located 68 nt downstream of the ORF 1a termination codon. ORF 2 (nt 4472 to 6619), which may be present also in the subgenonic RNA (Fig. 3), began with an initiation codon at nt 4571 and ended with a stop codon 308 bases upstream from the 3' end. ORF 2 was 2,148 nt in length and may encode a polypeptide of 683 aa.

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Schematic representation of the ANV genome. Open boxes, ORFs. The locations of three ORFs, predicted transmembrane helices (MB), protease (Pro), nuclear localization signal (NLS), ribosomal frameshift structure (RFS), RNA-dependent RNA polymerase (Pol), and stem-loop II-like motif (s2m) are indicated. Numbering is according to the ANV genonic sequence (accession no. AB033998).

View larger version (30K): [in this window] [in a new window]   FIG. 3.   RNA blot hybridization analysis. Total cytoplasmic RNA isolated from ANV-infected cells and uninfected cells at the indicated hours p.i. were resolved in a 1.0% agarose gel which contained 0.1% sodium dodecyl sulfate, transferred to a nylon membrane, and probed with DIG-labeled ANV probes (7FB-7RB, 5' end; 1F-1R, 3' end). The positions of the 28S and 18S rRNAs are indicated on the left; those of genonic (7.5-kb) and subgenonic (3.0-kb) RNAs are indicated on the right. cont, control.

RNA blot hybridization. By using a 3'-end probe in the Northern blot hybridization analysis, two RNA bands of 7.5 and 3.0 kb were identified in the ANV-infected cells at 16 and 24 h p.i. On the other hand, only one large RNA (7.5 kb) was observed with a 5'-end probe (Fig. 3).

Comparison of the sequence. Comparison of the nucleotide sequence and amino acid sequence of ANV with sequences of other positive-strand RNA viruses identified a region of similarity with HAst that included the putative RdRp, serine protease, and 3'-end motif (17, 18, 21, 32, 33, 48). The nucleotide sequences of the ANV genonic RNA of ORF 1a, 1b, and 2 were compared with those of the HAst (Table 3). Between ANV (G-4260 strain) and HAst, the genonic composition was very similar except for the 5'- and 3'-end NTRs, but the homologies of ORFs 1a, 1b, and 2 were not very high. The highest amino acid homology was found in the product of ORF 1b (41.9%) (Table 3). Although the homologies were low, the typical amino acid motifs of serine protease and RdRp encoded by ORF 1a and 1b, respectively, were conserved (Fig. 4) (21).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Amino acid sequence homology of the predicted functional domains of ANV (G-4260) with those of HAst 1 (A/88 Newcastle). (A) Putative 3C-like serine protease domains (ORF 1a). **, putative catalytic residues; !, residues implicated in substrate binding (16); *, identical residues; colons, similar residues. (B) Putative RNA-dependent RNA polymerase domains (ORF 1b). Consensus 1 shows amino acid residues that are conserved in at least 80% of the polymerases of supergroup I (16, 19). U, bulky aliphatic residue (I, L, M, or V); @, aromatic residue, (F, Y, or W); &, bulky hydrophobic residue (aliphatic or aromatic); ·, any residue. Residues conserved in the (putative) polymerases of all positive-strand RNA viruses of eukaryotes are in boldface. Distances between the aligned conserved motifs and from the protein termini are indicated.

View larger version (13K): [in this window] [in a new window]   FIG. 5.   Nucleotide sequence and predicted RNA secondary structure of the overlap region of ANV ORFs 1a and 1b. The deduced amino acid sequences encoded by ORF 1a, 1b, and 1a-1b surrounding the frameshift site are shown (17). The putative frameshift site (shifty heptanucleotide sequence) is underlined, and the termination codon of ORF 1a is boxed.

View larger version (7K): [in this window] [in a new window]   FIG. 6.   Phylogenetic relationship for ORF 1b (RdRp) and ORF 2 (structural polyprotein) of astroviruses. Amino acid sequences were analyzed using GENETYX-WIN, version 4 (Software Development Co., Ltd.). The phylogenetic trees were constructed by the unweighted pair group method by arithmetic averaging. The following nucleotide sequences were obtained from GenBank/EMBL/DDBJ: HAst 1, Z25771 and L23513; HAst 2, L13745; FAst (feline astrovirus), AF056197; PAst (porcine astrovirus), AB037272; TAst (turkey astrovirus), AF206663.

Morphology and physicochemical properties of ANV. In ultrathin sections, ANV particles were visible in the cytoplasm of infected cells as large aggregates (14). Negative-staining observation of purified ANV demonstrated that the particles were spherical and about 30 nm in diameter, but the five- or six-pointed star-like structure, a characteristic of the astroviruses (13, 26, 39), was not observed, although some empty particles were also observed (Fig. 7). The unstable character of ANV in CsCl solution was reproduced.

View larger version (161K): [in this window] [in a new window]   FIG. 7.   Electron micrograph of purified ANV (G-4260 strain) particles. Bar = 100 nm.

Infectivity in vitro of RNA transcribed from cDNA and cDNA clones of ANV (G-4260 strain) in CK and BHK cells. Beginning 48 h posttransfection (p.t.), ANV antigens were detected in the cytoplasm of the transfected CK cells by FA tests using anti-ANV chicken serum (Fig. 8). Typical ANV CPEs were observed at 48 h p.t. for RNA-transfected CK cells and at 72 h p.t. for cDNA-transfected CK cells. The culture supernatant contained infectious ANV at a titer of 10^6.5 50% tissue culture infective doses (TCID₅₀)/ml at 96 h after RNA transfection.

View larger version (106K): [in this window] [in a new window]   FIG. 8.   Immunofluorescence of CK (left) and BHK (right) cells transfected with in vitro-transcribed RNA from pANV-750 or a plasmid which can transcribe the full-length ANV cDNA under the control of a cytomegalovirus promoter. Fluorescing fine granules are disseminated in the cytoplasm of the cells. The viruses produced in these cells were transmissible to CK cells 96 h p.t. Magnification, ×360.

	DISCUSSION

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The genome structure of ANV, which is essential for determining the taxonomy of viruses, had not been shown since the first isolation in 1976 even though the virus had been believed to be a member of the family Picornaviridae by morphological (Fig. 7) and physicochemical analyses (31, 52). This study indicated that the ANV genome consists of approximately 7,000 nt with three ORFs, ORF 1a, 1b, and 2 (Fig. 2). ORF 1a encoded a 3C-like serine protease motif, whereas the ORF 1b encoded a viral RdRp motif, and a ribosomal frameshift motif was also present between the two ORFs. On the other hand, ORF 2 of ANV, which may encode the capsid precursor polyprotein, encoded a product with 26% amino acid homology to those of HAst (17, 34, 48). As shown for the HAst, ORF 2 of ANV is likely expressed from subgenonic-size RNA (29, 34), which was detected in the ANV-infected cells, as well as genonic-size RNA (Fig. 3). The genome organization of ANV is apparently identical to that of HAst (Fig. 2) (3). The sequence analyses clearly demonstrated that ANV is not a Picornavirus but a member of the family Astroviridae (31, 33, 35).

ANV differs from previously described mammalian astroviruses in that trypsin is not required for growth in tissue culture (52). Trypsin-dependent replication of astroviruses in vitro is a very specific feature (3, 42, 47, 50). Virus particles produced in the absence of trypsin are minimally infectious. Monroe et al. demonstrated that the trypsin-free virus particles were composed of the precursor capsid protein (34). Bass and Qiu clearly proved that conversion of the large precursor protein (79 kDa) to three small mature capsid proteins (34, 29, and 26 kDa) correlated with enhanced infectivity by trypsin treatment (1). It is not clear why ANV does not require trypsin to replicate in CK cell culture in spite of being an astrovirus. One of the reasons might be that the CK cell is not a lined cell but a primary culture cell.

Picornaviruses can usually be purified with CsCl ultracentrifugation, but ANV cannot. The unstable property of ANV in CsCl solution was also described for HAst 1 but not for HAst 2 (29). Some animal astroviruses of bovine, ovine, porcine, feline, turkey, and duck origin have already been reported, and some of them are classified as Astroviridae mainly from their morphological properties (11, 12, 33, 40, 42, 50; R. E. Gough, M. S. Collins, E. Borland, and L. F. Keymer, Letter, Vet. Rec. 114:279, 1984). ANV particles did not show the typical star-like motif (Fig. 7); however, it is known that only minor populations of virions exhibit this star-like morphology, and they are visualized in stool specimens as star-like particles (26, 33, 39).

ANV is the first astrovirus which was not identified by the morphological character but by genonic analyses. The first AUG codon of ORF 1a remains to be identified because there are not typical motifs. The 5'-end NTR of the ANV G-4260 strain (13 nt) was short if the virus was using the first methionine (GXXAUGG) (22) in comparison with those of HAst (80 to 82 nt). When the virus uses the first methionine (AXXAUGG) (22), ORF 2 may encode a capsid protein precursor of 683 aa with a predicted molecular mass of 73.9 kDa, which is smaller than those of HAst (88 to 90 kDa) (17, 34). On the other hand, although the 3'-end NTR of ANV was more than three or four times the lengths of those of other astroviruses (Table 2), the common RNA motif (35 nt) of astroviruses was identified at nt 6707 to 6739 (90.9% homology) (18, 32). This common RNA motif is also found in the 3' ends of the genomes of avian IBV and an equine rhinovirus (18). However, the position of the motif, which was named a stem-loop II-like motif (s2m) (Fig. 2), was different somehow in different viruses. In HAst, this motif contains the stop codon of ORF (18). IBV is a coronavirus with a 27.6-kb plus-strand RNA genome containing a 0.3- to 0.5-kb 3' NTR and a poly(A) tail (49). Different strains have different tissue tropisms, infecting respiratory, reproductive, and gastrointestinal organs as well as the kidneys of chickens (44, 45). We also suggest that this conserved RNA motif in these different viruses might have resulted from a recombinational event during coinfection, because both ANV and IBV replicate in the chicken kidneys and gut (14, 18, 45). Of interest, astrovirus-like particles have been reported (Gough et al., Vet. Rec. 114:279) in association with fatal hepatitis in ducklings, suggesting a possible hepatic tropism for this virus.

Our study showed that ANV was genetically distinct from other astroviruses (Fig. 6). The RdRp polyprotein sequence of ANV (ORF 1b) had a 41.9% overall amino acid homology with that of HAst, while the polyprotein sequences encoded by ANV ORF 1a and 2 had 20 and 26% amino acid homology, respectively (Table 3). Turkey astrovirus was the closest virus to ANV. ORF 1a, 1b, and 2 of ANV encoded products with 29.9, 53.8, and 32.3% overall amino acid homology to those of turkey astrovirus (AF206663). Although the antigenic studies remain, from the comparison of the sequence and biological properties of these two viruses, they seem different (46). In the current taxonomy of picornaviruses, an amino acid homology of the RdRp lower than 60% is one of the important parameters to assign a new genus along with the genome structure and the biological character (19, 37).

The present findings strongly support the classification of ANV as a new genus of the family Astroviridae, and the virus group of Astroviridae can cause nephritis in chickens, in addition to gastroenteritis.

The ANV infectious cDNA clone described here should prove useful for defining the minimal and optimal requirements for the use of astrovirus groups as an expression vector and for developing a packaging system for vector RNA (10), and this cDNA clone would greatly facilitate and enhance studies on the replication strategy and the pathogenesis of the astroviruses in vitro and in the original host.