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
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this paper are AB084788 and AB097152AB097166.
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INTRODUCTION |
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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.
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METHODS |
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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)1218 (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 AB097152AB097166.
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.
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RESULTS |
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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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DISCUSSION |
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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 stemloop 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 stemloop 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.
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Received 1 April 2003;
accepted 6 August 2003.