Isolation and characterization of a new species of kobuvirus associated with cattle

Teruo Yamashita, Miyabi Ito, Yuka Kabashima, Hideaki Tsuzuki, Akira Fujiura and Kenji Sakae

Department of Microbiology, Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsujimachi, Kita-ku, Nagoya, Aichi 4628576, Japan

Correspondence
Teruo Yamashita
tyamashita{at}hi-ho.ne.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A cytopathic agent was isolated using Vero cells from the culture medium of HeLa cells that had been used for more than 30 years in our laboratory. This agent, termed U-1 strain, was serially passed in Vero cells with distinct CPE. Particles of U-1 strain negatively stained with phosphotungstic acid exhibited a distinct surface that resembled Aichi virus. The RNA genome of U-1 strain comprises 8374 nt, with a genome organization analogous to that of picornaviruses. Possible cleavage sites of the large ORF, which encoded a leader protein prior to the capsid protein region, were assigned following amino acid alignment with Aichi virus. The virus sequence had 33 and 75 % amino acid identity with the Aichi virus VP1 and 3D regions, respectively, but no more than 23 and 36 % with those of the prototype strains of other Picornaviridae. The dendrogram based on the P1, P2 and P3 proteins indicated that U-1 strain is genetically included in the genus Kobuvirus but is distinct from Aichi virus. Of 72 cattle sera, 43 (59·7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 8 or more. However, sera from 190 humans, 242 monkeys, 139 pigs, 5 horses, 22 dogs and 9 cats did not neutralize U-1 strain at a 1 : 4 dilution. RNA corresponding to U-1 strain was detected in 12 (16·7 %) of 72 faecal samples from cattle by RT-PCR. These results indicated that U-1 strain, suspected to be a contaminant from calf sera, is a new species of the genus Kobuvirus, now termed bovine kobuvirus.

The GenBank accession numbers of the sequences reported in this paper are AB084788 and AB097152AB097166.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Picornaviruses consist of a naked capsid that surrounds a core of ssRNA. Hydrated native particles are 30 nm in diameter but vary from 22 to 30 nm in electron micrographs, which reveal the virion as a smooth sphere. Picornaviridae comprises nine genera: Enterovirus, Rhinovirus, Aphthovirus, Cardiovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus and Teschovirus (King et al., 2000; Pringle, 1999). Aichi virus is a species of the genus Kobuvirus (Stanway et al., 2002), with a rough virion surface, and has been isolated from patients with gastroenteritis (Yamashita et al., 1991). The Aichi virus (A846/88) genome is 8280 nt long and encodes a single polyprotein of 2432 aa. This polyprotein has a clear similarity with other picornaviruses. However, there are some characteristic regions of the Aichi virus genome organization. For example, Aichi virus has an L protein consisting of 170 aa and only three structural proteins, VP0, VP3 and VP1 (Yamashita et al., 1998). Hughes & Stanway (2000) observed conserved motifs, previously unrecognized, in the Aichi virus, human parechovirus (HPeV) and avian encephalomyelitis virus (AEV) 2A proteins.

Recently, the genome sequences of AEV and Ljungan virus from bank voles have been determined and revealed to be members of the Picornaviridae and most closely related to hepatitis A virus and HPeV, respectively (Marvil et al., 1999; Niklasson et al., 1999). Thus, most picornavirus genera consist of two or more species. We have studied 17 virus isolates of human patients with gastroenteritis and determined these to be Aichi viruses via a neutralization test with Aichi virus (A846/88) antisera (Yamashita et al., 1991, 1993, 1995). To date, Aichi virus is the only species of the genus Kobuvirus.

Using Vero cells, we detected recently a cytopathic agent in the culture medium of HeLa cells. This agent could not be neutralized by Aichi virus antisera but contained some features similar to Aichi virus and reacted with antibody raised to Aichi virus by ELISA. In this study, we demonstrate by sequencing the entire genome that this agent is a new species of the genus Kobuvirus. Moreover, an epidemiological study performed suggested that our HeLa cells were contaminated with this virus, termed U-1 strain, which had originated presumably from cattle sera.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HeLa cells.
HeLa cells were provided to our laboratory by H. Sunaga (Nagoya University School of Medicine, Nagoya, Japan) in the 1960s. The unique colony name is not available. At that time, HeLa cells were cultivated using MEM containing 10 % calf serum. From the 1980s, however, foetal calf serum (FCS) was used for the medium instead of calf serum. HeLa cl.2 cells (ATCC) were purchased from Dainippon Seiyaku and cultivated in plastic flasks using MEM with 10 % FCS for virus isolation tests.

Isolation of U-1 strain and preparation of antisera.
Medium from cultivated HeLa cells was inoculated onto Vero cells. CPE was confirmed only in the sample medium cultivated with the HeLa cells provided from Nagoya University. The cytopathogenic agent was plaque-cloned and termed U-1 strain. Serological, biochemical and biophysical analyses were performed with U-1 strain, as described previously (Yamashita et al., 1991). Briefly, U-1 strain was incubated in 10 % chloroform for 10 min at room temperature and in MEM (pH 3·5) for 3 h at room temperature to evaluate its stability in organic solvents and under acidic conditions, respectively. The type of U-1 strain nucleic acid was determined by examining the effects of 10-4·5 M 5-iodo-2'-deoxyuridine (IUDR), a DNA virus inhibitor of virus replication. U-1 strain cultivated in Vero cells was purified using caesium chloride and sucrose density gradient centrifugation. Aichi virus (A846/88 strain) and poliovirus type 1 (PV-1, Sabin strain) were also grown and purified in the same manner. Purified viruses were examined under electron microscopy and by SDS-PAGE and were also used for the preparation of antisera and for ELISA. Immune sera were obtained from guinea pigs that were inoculated experimentally with Aichi virus (A846/88) and U-1 strain, respectively.

cDNA synthesis and cloning.
The complete nucleotide sequence of purified U-1 strain was determined as described previously (Yamashita et al., 1998), with modifications. Briefly, virion RNA was extracted using TRIzol (Invitrogen), following the instructions of the manufacturer. AMV reverse transcriptase (Promega) was used to create ss cDNA, with oligo(dT)12–18 (Promega) and random six-residue primers (Takara). ds cDNA preparation and cloning into pBR322 and pUC19 were done as described previously (Supanaranond et al., 1992; Takeda, 1989).

U-1 strain sequence-specific oligonucleotides were designed on the basis of sequences near the ends of the cloned cDNAs and were used for PCR. Oligo(dT)33 was used for the extreme 3' end of the genome. Six clones in a pGEM-T vector (Promega) background were obtained and sequenced to bridge the gaps between the pUC19 cDNA clones. The clones of the extreme 5' end of the genome were obtained using the 5' RACE kit (Roche), as described elsewhere (Sasaki et al., 2001).

Stool and serum samples.
Stool and serum samples from 2- to 4-year-old calves were obtained, together with pig serum samples, from slaughterhouses in the Aichi Prefecture. Human serum samples were obtained from the Japanese Red Cross. Horse serum samples were purchased from a market for laboratory use. Dog and cat serum samples were obtained from T. Kato, Kato Veterinary Hospital, Aichi Prefecture, Japan. Monkey serum samples were obtained from K. Asaoka, Kyoto University Primate Research Center, Kyoto, Japan. Calf stool samples were prepared as 10 % homogenates in PBS and centrifuged at 10 000 g for 20 min. Resultant supernatants were inoculated onto Vero cells and used for RT-PCR, as described below.

ELISAs.
ELISAs was used to identify the reactivity of U-1 strain against Aichi virus and PV-1. ELISA plates (Nunc) were coated with 0·2 mg purified U-1 strain, Aichi virus or PV-1 per well and blocked with PBS/T (0·05 % Tween 20 and PBS) and 2 % BSA (Sigma). To each well of the plates were added a 100-fold or higher dilution of anti-U-1 strain or anti-Aichi virus serum. Plates were incubated overnight at 4 °C. After washing with PBS/T, peroxidase-labelled rabbit anti-guinea pig IgG (Zymed) in PBS/T with 1 % BSA was added to each well and incubated for 2 h at 37 °C. o-Phenylenediamine (Sigma) was used for colour development. After 30 min at room temperature, the reaction was stopped by addition of 4 M H2SO4. Absorbance readings were taken at 490 nm using a plate spectrophotometer (Corona Electric). Endpoint titres of the sera were defined as A490>0·15 (greater than three times the negative control well – without virus antigen).

RT-PCR.
Primers for RT-PCR were designed based on the sequences of the Aichi virus and U-1 strain genomes. Oligonucleotide primer sequences were selected as follows: 10f (sense, 5'-GATGCTCCTCGGTGGTCTCA-3'; nt 7357) and 10r (anti-sense, 5'-GTCGGGGTCCATCACAGGGT-3'; nt 7987), which amplifies a 631 bp region of the 3D protein. RNA extraction from faecal samples was performed as described previously (Yamashita et al., 2000). In brief, faecal extracts were centrifuged at 10 000 g for 20 min and the supernatant was collected for RT-PCR. As described by Jiang et al. (1992), 0·2 ml faecal extract was mixed with 0·1 ml 24 % polyethylene glycol-6000 and 1·5 M NaCl, stored at 4 °C overnight and centrifuged at 3000 g for 20 min. The pellet was suspended in 0·1 ml RNase-free water for RT-PCR. Virus RNA was extracted using TRIzol followed by isopropanol precipitation. Nucleic acid was suspended in reverse transcription mixture (Invitrogen) containing 1 µM oligo(dT)15 (Promega) and 1 µM random primer (Takara) and incubated for 60 min at 37 °C. PCR mixtures containing primers were added directly to each of the reverse transcription mixtures and amplification was performed in a Thermal Cycler 9600 (Cetus, Perkin-Elmer) for 40 cycles (95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min). Analysis of the amplification product was performed by agarose minigel electrophoresis and confirmed as a distinct band by staining with ethidium bromide. Following RT-PCR, amplified products from positive faecal samples were purified by phenol/chloroform extraction. Purified RT-PCR products were then precipitated with ethanol and pelleted DNA was suspended in 10 mM Tris/HCl buffer (pH 8·5) and introduced into a pGEM-T vector (Promega). Aichi virus isolates (A846/89, A1156/87, M166/91 and P766/90) from patients with gastroenteritis (Yamashita et al., 2000) were grown in Vero cells and used as samples for RT-PCR and DNA sequencing.

DNA sequencing and analyses.
Inserts containing identified cDNA plasmid clones were used to determine nucleotide sequences using a SequiTherm LongRead Cycle Sequencing kit (Epicentre Technology) and an automated DNA sequencer (Model 4000, Li-Cor). The nucleotide sequence was analysed at least twice in both directions. The complete U-1 strain sequence has been submitted to the DDBJ/EMBL/GenBank databases under accession no. AB084788. The sequences amplified using the primers 10f and 10r have also been deposited in the databases under accession nos AB097152–AB097166.

Sequence comparisons between U-1 strain and Aichi virus (A846/88, accession no. AB010145 and AB040749) were made using the GCG sequence analysis package. A dendrogram was constructed using UPGMA (unweighted pair group method with averages) in the same package. The secondary structures of the 5'- and 3'-terminal nucleotides were predicted using the MFOLD program (Mathews et al., 1999). The following nucleotide sequences were also obtained from DDBJ/EMBL/GenBank database: avian encephalomyelitis-like virus (AEV), AJ225173; bovine enterovirus type 1 (BEV-1), D00214; coxsackievirus A16 (CV-A16), U05876; coxsackievirus A21 (CV-A21), D00538; coxsackievirus B3 (CV-B3), M16572; encephalomyocarditis virus (EMCV), M81861; enterovirus type 70 (EV-70), D00820; equine rhinitis A virus (ERAV), L43052; equine rhinitis B virus (ERBV), X96871; foot-and-mouth disease virus type O (FMDV-O), X00871; hepatitis A virus (HAV), M14707; human parechovirus type 1 (HPeV-1), L02971; human rhinovirus type 2 (HRV-2), X02316; human rhinovirus type 14 (HRV-14), K01087; Ljungan virus (LV), AF327920; poliovirus type 1 (PV-1), J02281; porcine enterovirus type 9 (PEV-9), Y14459; porcine teschovirus (PTV), AJ011380; and Theiler's murine encephalomyelitis virus (TMEV), M20301.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Biological properties of U-1 strain
U-1 strain was serially passaged in Vero cells, resulting in distinct CPE, and was titrated successfully using Vero cells grown in microplates (4x104–4x105 TCID50 ml-1). The addition of IUDR (10-4 M) to the Vero cell culture at the time of infection did not prevent CPE produced by U-1 strain. The results indicated that the nucleic acid type was RNA. It was stable to treatment with chloroform and acid (pH 3·5). Distinct virus particles could be seen following negative-staining for 2 min with phosphotungstic acid (pH 7·2) and had an average diameter of 30 nm, which resembles Aichi virus but is different from PV-1 (Fig. 1).



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Fig. 1. Electron micrograph of Aichi virus, U-1 strain and PV-1 negatively stained with 2 % phosphotungstic acid (pH 7·2) for 2 min.

 
Reactivity of U-1 strain with EV, HPeV and Aichi virus
U-1 strain (4x103 TCID50 ml-1) could not be neutralized by 20 units of antibody against 64 prototype strains of EV, two prototype strains of HPeV and Aichi virus. U-1 strain antibody at a titre of 1 : 1000 could not neutralize these viruses. Purified PV-1, Aichi virus and U-1 strain (all at 0·2 mg ml-1) were used to confirm reactivity with anti-Aichi virus and anti-U-1 strain sera using ELISA. Anti-U-1 strain at a titre of 1 : 16 000 did not react with Aichi virus and PV-1. However, anti-Aichi virus sera at a titre of 1 : 80 000 did react with U-1 strain at a titre of 1 : 40 000. In contrast, it did not react with PV-1 (Table 1). This result raised the possibility that there is a group-specific antigen between Aichi virus and U-1 strain.


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Table 1. Antigenic reactivity between U-1 strain and Aichi virus, as determined by ELISA

 
Complete nucleotide sequence of U-1 strain
A total of 13 overlapping cDNA clones spanning the entire genome of U-1 strain were obtained and their nucleotide sequences determined. The RNA genome of U-1 strain consists of 8374 nt, excluding the poly(A) tail. A large ORF of 7392 nt, which encodes a potential polyprotein precursor of 2464 aa, was found, preceded at the 5' end by 808 nt, and followed at the 3' end by 174 nt and a poly(A) tail. The base composition was found to be 20·2 % A, 21·7 % G, 32·9 % C and 25·2 % U. The U-1 strain polyprotein sequence was analysed for potential cleavage sites based on an alignment with the Aichi virus sequence. Possible cleavage sites for VP3/VP1, VP1/2A and 3B/3C were Q/A (Q/T in Aichi virus), Q/C (Q/G in Aichi virus) and Q/A (Q/G in Aichi virus), respectively. Other cleavage sites were found to be identical to those in Aichi virus (Table 2). In a previous study (Yamashita et al., 1998), the first 32 nt of the Aichi virus genome were not identified and the published sequence (AB010145) exhibited 26 sequence differences from a sequence of Aichi virus (AB040749) published by Sasaki et al. (2001). These differences included ones that cause alterations in the 28 amino acid sequences in the VP0 region. The deduced amino acid sequence of the U-1 strain VP0 region was more analogous to that of the newly published ones than the earlier one. Therefore, U-1 strain RNA and polyprotein sequences were compared with the latter Aichi virus sequence (accession no. AB040749), as shown in Table 2.


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Table 2. Comparisons of RNA and amino acids between U-1 strain and Aichi virus

 
Analysis of U-1 strain UTRs
The U-1 strain 5' UTR was 64 bases longer than that of the Aichi virus 5' UTR and its sequence identity was 49·3 % (Table 2). The predicted secondary structure of the 5'-terminal 116 nt consisted of three stem–loop domains. In particular, the first 50 bases, which comprised a stem–loop structure, was different by only one base from the Aichi virus stem–loop and the structure was found to be identical. The pyrimidine-rich tract was present between nt 765 and 771 and this was followed by the initiator methionine (AUG) at position 37. The ‘spacer’ of the U-1 strain was 26 nt longer than that of Aichi virus, which is 11 nt. An additional 89 nt were inserted before the pyrimidine-rich tract of U-1 strain (Fig. 2) in comparison with Aichi virus. The secondary structures of the internal ribosome entry site (IRES) are not known. The U-1 strain 3' UTR was 63 bases shorter than that of the Aichi virus 3' UTR but for the remainder the identity was 47·7 % (Table 2). The predicted secondary structure of the 3' UTR of U-1 strain consisted of three ds hairpin stems but the secondary structure of the 3' UTR of Aichi virus could not be defined (Yamashita et al., 1998).



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Fig. 2. Alignment of the 5' UTRs of U-1 strain and Aichi virus. Arrows indicate the positions of three stem–loop structures. AiV, Aichi virus; Yn, pyrimidine-rich tract; Xm, nonconserved sequence; AUG, initiator methionine.

 
Analysis of U-1 strain coding regions
U-1 strain has a leader protein comprising 187 aa, which was 17 aa longer than Aichi virus, with an identity of 31 %. PAGE analysis of purified virus revealed three capsid proteins with molecular masses of 39, 29 and 22 kDa. Comparison of these proteins with those of Aichi virus and the molecular mass calculated for amino acids of the U-1 strain structural proteins indicated that the 39, 29 and 22 kDa polyproteins were VP0, VP1 and VP3, respectively (Fig. 3). The percentage identity between the U-1 strain and Aichi virus VP0 proteins (57·5 % amino acid identity) was similar to that comparing the VP2 sequences and higher than that of VP1 regions (33·2 %). VP1 sequences varied in length: 253 aa in Aichi virus and 267 aa in U-1 strain (Table 2).



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Fig. 3. SDS-PAGE of Aichi virus (AiV) and U-1 strain. The molecular masses (kDa) of VP0, VP1 and VP3 are shown on the right.

 
The amino acid identity of the P2 and P3 proteins (nonstructural proteins) varied from 44·9 % (3A) to 74·8 % (3D) between the U-1 strain and Aichi virus (Table 2). The amino acid sequence of the 2A region of U-1 strain had 57·4 % identity with Aichi virus and this conserved motif consisted of H-box/NC proteins and a transmembrane domain, as described elsewhere (Hughes & Stanway, 2000). Amino acid sequences of the 2C region were well aligned with Aichi virus, including the highly conserved motif GPPGTGKS, which is the nucleotide-binding domain of the putative picornavirus helicase. A catalytic triad formed by histidine, glutamate and cysteine in 3C was seen in U-1 strain at the same amino acid positions as in Aichi virus. The amino acid sequence of the 3D region aligned well, including the KDELR, YGDD and FLKR motifs of the RNA-dependent RNA polymerase.

Phylogenetic analysis
The relationships between the proteins of U-1 strain and those of other picornaviruses were examined by UPGMA, using amino acid multi-alignment of the P1, P2 and P3 regions. The dendrograms based on the P1, P2 and P3 proteins are depicted in Fig. 4. This confirmed that U-1 strain was clearly more closely related to Aichi virus than to any other picornaviruses, but the evolutionary distance between U-1 strain and Aichi virus was equivalent to that between representative species in each genus of the Picornaviridae.



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Fig. 4. Relationships between Aichi virus and other picornaviruses based on deduced amino acid differences of the P1, P2 and P3 proteins. The dendrograms were generated by evolutionary distances, as computed by UPGMA.

 
Prevalence of U-1 strain antibodies
Of 72 cattle sera, 43 (59·7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 16 or more. However, 190 human, 242 monkey, 139 pig, 5 horse, 22 dog and 9 cat sera did not neutralize U-1 strain at a serum dilution of 1 : 8. In contrast, antibody to Aichi virus was detected only in human samples (Table 3).


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Table 3. Prevalence of U-1 strain and Aichi virus antibodies in different species

 
Detection of U-1 strain-like RNA in cattle stool samples
A total of 72 faecal specimens from healthy cattle were examined by passage in Vero cells and RT-PCR using the primer pair 10f and 10r. No U-1 strain-like virus could be isolated from the Vero cells but virus-specific RNA could be detected in 12 (16·7 %) of 72 samples using RT-PCR directly from faecal material. (The farms from which RNA-positive cattle were raised were located in different areas of the Aichi Prefecture.) The primer pair 10f and 10r could amplify products from U-1 strain and four Aichi virus isolates obtained from human faecal samples described elsewhere (Yamashita et al., 1995). The sequences of the RNAs amplified from 12 cattle had approximately 90 % identity with U-1 strain and 60 % with the four Aichi virus isolates.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study reports on the biological and serological properties and complete nucleotide sequence and genetic organization of a cytopathogenic agent, U-1 strain, isolated from HeLa cells on Vero cells. Biological and phylogenetic analyses revealed that this virus bears many similarities to Aichi virus and can be considered a new member of the genus Kobuvirus. Furthermore, epidemiological studies showing the prevalence of neutralizing antibody in some animals and the detection of RT-PCR products from cattle faeces warrants that this virus be a new species of the genus Kobuvirus, called bovine kobuvirus.

The identity of the 5' UTRs between U-1 strain and Aichi virus was low. However, the first 50 bases differed by only one base and the secondary structure was identical. The 42 nt at the 5' end of the genome formed a stable stem–loop structure and plays an essential role in the formation of virus particles as well as in RNA replication (Sasaki et al., 2001). Our results strongly support this, suggesting the importance of this region and revealed that the secondary structure of this region is distinctive to this genus. The location of the pyrimidine tract and initiator methionine suggests that Aichi virus has an IRES that is most similar to type II IRES sequences (Yamashita et al., 1998). When we compared the sequences of the 5' UTRs of U-1 strain and Aichi virus, significant differences surrounding the pyrimidine tract could be identified. Therefore, the predicted secondary structure of the IRES consisting of stem–loop structures may be preserved in this genus, although the secondary structures of Aichi virus and U-1 strain IRES sequences are not known.

The major differences of Aichi virus from other picornaviruses are found in the coding region of the L protein, the absence of a VP0 cleavage site and a distinct form of the 2A protein (Stanway et al., 2002). U-1 strain also exhibited this feature. When we compared amino acid identities between U-1 strain and Aichi virus, the percentage identity in the L protein (31·0 %) was lower than the 3A (44·9 %) or 3B (43·3 %) proteins. Aphthoviruses and cardioviruses also encode an L protein. The cleavage activity of the aphthovirus (FMDV) L protein has been well characterized (Piccone et al., 1995; Strebel & Beck, 1986) and the cardiovirus TMEV L protein has been shown to be a zinc-binding protein that may play a role in restricting host cell growth (Chen et al., 1995). The L proteins of U-1 strain and Aichi virus exhibited relatively low similarity to each other and no sequence identity to the L protein of aphthoviruses or cardioviruses. As a result, we cannot currently deduce a function for the L protein of U-1 strain and Aichi virus.

The Aichi virus VP0 protein has been shown to strongly react with convalescent-phase serum from patients (Yamashita et al., 1991); therefore, it is probably exposed on the surface of the virions. However, the percentage identity of the VP1 protein between U-1 strain and Aichi virus was lower than that of the VP0 region. VP1 is the most exposed and immunodominant of the picornavirus capsid proteins (Rossmann et al., 1985) and in enteroviruses, VP1 sequences correlate with neutralization type (Oberste et al., 1999). Our results parallel this and suggest that the VP1 protein of kobuvirus was the most variable of the structural proteins.

The protein encoded at the 2A locus differs dramatically among picornaviruses and several distinct forms have been identified (Bazan & Fletterick, 1988; Donnelly et al., 1997; Ryan & Drew, 1994; Yu & Lloyd, 1992). It has been reported that the 2A protein of Aichi virus as well as HPeV and AEV contain conserved motifs (H-box/NC) that are characteristic of a family of cellular proteins involved in the control of cell proliferation (Hughes & Stanway, 2000). The 2A protein of U-1 strain was similar to Aichi virus (57·4 % amino acid identity) and possessed these H-box/NC proteins. This percentage identity is higher than that of the 3C protein (47·9 %) and suggests that the 2A protein of kobuvirus may perform an important mechanism.

An atypical genome and codon base composition of Aichi virus has been pointed out elsewhere (Palmenberg & Sgro, 2002). The pyrimidine content (38 % C and 24 % U) of Aichi virus is higher than that of other picornaviruses. The triplet assignment in the standard genetic code is not random and the average picornavirus ratios for A : G : C : U are 30 : 31 : 19 : 20 (SD=4). Aichi virus, however, has a much higher than average C composition (C=28 %). In this study, 58 % of the U-1 strain base count was pyrimidine (33 % C and 25 % U). In the first codon base, the ratio of the U-1 strain base count was 23 : 32 : 25 : 21 for A : G : C : U. The high C composition in the genome is suspected to be a typical skew of kobuviruses.

The prevalence of the antibody to U-1 strain and positive results of RT-PCR in healthy cattle revealed that U-1 strain-like viruses are common between these domestic animals and are excreted in the faeces. Our HeLa cells were suspected to be infected with U-1 strain through a culture medium supplemented with calf serum, which had been possibly polluted with faeces. This discovery was not readily apparent, since it had grown in HeLa cells without any CPE. This was not necessarily surprising, because several species of picornaviruses have been identified as persistent infections in vitro (de la Torre et al., 1985; Gibson & Righthand, 1985; Matteucci et al., 1985; Roos et al., 1982; Vallbracht et al., 1984). Our results highlight the fact that HeLa cells kept in other laboratories may also have been contaminated with this virus.

BEV strains are well known to be endemic in cattle in many regions of the world. Although an infection of BEV is known to be asymptomatic, it can also been associated with diarrhoea and, on occasion, abortion (Ley et al., 2002). The prevalence of anti-Aichi virus antibodies in man also suggests that there are likely to be many asymptomatic infections (Yamashita et al., 1993, 1995). However, isolates of Aichi virus have been found only in patients with gastroenteritis. In this study, we detected kobuvirus-specific RNA in 12 (16·7 %) of 72 faecal samples from apparently healthy cattle by RT-PCR. These findings suggested that U-1 strain-like virus infections may be typically asymptomatic in cattle. However, we were not able to isolate a U-1 strain-like virus with Vero cells from the faeces of healthy cattle positive for kobuvirus-specific RNA. Like Aichi virus, this virus may be isolated only from symptomatic cattle. More epidemiological studies are required regarding the significance of this virus as a causative agent of some diseases of cattle. The development of RT-PCR for kobuvirus should prove useful for this study. To isolate U-1 strain-like viruses, another type of cell, such as bovine cells, may be required.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 1 April 2003; accepted 6 August 2003.