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Animal: RNA Viruses |
Department of Virology, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan1
Tsukuba Life Science Center, The Institute of Physical & Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan2
Author for correspondence: Yoshihiro Kaku. Fax +81 298 38 7880. e-mail kaku{at}niah.affrc.go.jp
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Abstract |
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 TOP Abstract IntroductionMethodsResults and DiscussionReferences |
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Introduction |
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TOPAbstractIntroductionMethodsResults and DiscussionReferences |
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). Moreover, echoviruses 22 and 23 were defined as a sixth genus, Parechovirus (Mayo & Pringle, 1998 ), and renamed human parechoviruses 1 and 2 (HPeV-1 and -2). Recently, three new genera were approved at the 11th International Congress of Virology in 1999, Erbovirus, Kobuvirus and Teschovirus, which includes porcine teschovirus (PTV), previously named porcine enterovirus 1 (Pringle, 1999 ).
Porcine enteroviruses (PEVs) belonging to the genus Enterovirus have been divided into 11 serotypes (PEV-1 to -11; ‘ICTV classification’ in Table 1) based upon the virus-neutralization test (Knowles et al., 1979 ). In addition, Japanese isolates have been classified independently into 10 serotypes (PEV-J1 to -J10; ‘Japanese classification’ in Table 1), some of which overlap PEV-1 to -11 (Honda et al., 1990 ). Moreover, two new serotypes were identified recently in Germany (Auerbach et al., 1994 ). Consequently, at least 15 serotypes have been identified to date. Furthermore, these serotypes have also been divided into three groups (I, II and III) based upon the type of cytopathic effect (CPE) produced in pig kidney cells, physico-chemical properties and different cell culture host ranges (Honda et al., 1990 ; Knowles et al., 1979 ; Rasmussen, 1969 ) (Table 1).
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; Harding et al., 1957 ) and were designated Teschen strain and Talfan strain. In addition, strains of various serotypes have been isolated from pigs showing polioencephalomyelitis (Kasza, 1965 ), enteric disease (Izawa et al., 1962 ), pneumonia (Meyer et al., 1966 ) and a lack of symptoms (Izawa et al., 1962 ). This situation has obscured the relationship between virulence and serotypes/CPE types. Although an alternative classification based upon genetic information has been required, few molecular studies have been performed until recently. In 1999, the partial genomic sequences of two PEV-1 strains were determined. One was the majority of the sequence, excluding the 5' non-translated region (5'-NTR), of strain F65 isolated from a pig showing no clinical symptoms (Doherty et al., 1999 ), and the other was the sequence of the RNA-dependent RNA polymerase (RdRp) of the Talfan strain (Kaku et al., 1999 ). Phylogenetic analyses indicated that these viruses were genetically distinct from other picornaviruses, in contrast to the current taxonomy. As a result, PEV-1 was reassigned to a new genus, Teschovirus, and renamed PTV. However, the total reclassification of PEVs still remains to be done due to the lack of the genetic information on other PEV serotype strains. In this paper, Talfan, as the reference strain for PTV, and PEV strains of most serotypes were analysed genetically. Firstly, the majority of the nucleotide and amino acid sequence of Talfan was determined, except for the 5'-NTR. The sequence was compared with the sequences of other picornaviruses and was utilized to design primers for the ensuing analysis. Secondly, we investigated the genetic diversity among PTVs and PEVs, analysing the sequences of RT–PCR and 3'-RACE products, which were produced by using primers based on Talfan and other available PEV sequences. Finally, the total genetic reclassification of PTVs and PEVs is discussed.
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Methods |
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TOPAbstractIntroductionMethodsResults and DiscussionReferences |
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Virus strains.
). All strains were grown in IB-RS-2 cells derived from porcine kidney.
Sequence determination of the Talfan genome.
PTV Talfan was purified by the method of Inoue et al. (1989) . Genomic RNA extracted from purified virions was converted into cDNA with an oligo(dT) primer and random primers and cloned into 
ZAP II vector as described previously (Kaku et al., 1999 ). All clones were screened by hybridization with a [32P]dCTP-labelled virus genomic probe. Positive clones were sequenced by the dideoxy method using a DNA sequencing kit and an ABI 373 DNA sequencer (PE Applied Biosystems). Intervening sequences were obtained by analysis of RT–PCR fragments.
RT–PCR.
Genomic RNA of all strains was isolated from cultured medium using Sepasol RNA I (Nakalai Tesque). RT–PCR primers were designed to amplify the conserved region (Koonin & Dolja, 1993 ) of RdRp and the region encoding the N terminus of the capsid protein VP2, based on the sequences of three virus strains; Talfan, PEV-8/V13 and PEV-9/UKG410/73. These strains were selected from CPE types I, II and III, respectively. The sequences of PEV-8/V13 and PEV-9/UKG410/73 were obtained from the database (accession numbers AJ001391 and Y14459, respectively) except for the VP2 region of V13, which was obtained from a cDNA library. The sequences of the primers used and their locations are shown in Table 2. All reactions were performed with a TaKaRa RNA PCR kit according to the manufacturer’s manual. For reverse transcription, reactions were carried out at 42 °C for 30 min and the enzyme was inactivated at 95 °C for 5 min. For PCR, the reactions were amplified through 30 cycles by using a GeneAmp PCR System 9600 thermal cycler (PE Applied Biosystems). Denaturation was carried out at 94 °C for 1 min, primer annealing at 51 °C for 1 min and elongation at 72 °C for 1 min in all reactions. PCR amplicons were electrophoresed in 1·0% agarose gel. If several DNA bands were detected, an adequate band was cut out and extracted from the gel by using GenElute agarose spin columns (SUPELCO). All products were ligated into the cloning vector pCR 2.1-TOPO by using the TOPO TA cloning kit (Invitrogen) and sequenced as described above.
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3'-RACE.. All the reactions were performed using the TaKaRa RNA PCR kit. Reverse transcription was performed by using the oligo(dT)-adaptor primer as follows: 30 °C for 10 min, 50 °C for 30 min, 95 °C for 2 min and 5 °C for 5 min. For PCR, the reactions were amplified through 30 cycles using M13 primer M4 and strain-specific primers. Denaturation was carried out at 94 °C for 30 s, elongation at 72 °C for 30 s and primer annealing at 51 °C for 30 s in all reactions. All products were cloned and sequenced as described above.
Genetic analysis.
The sequence data were assembled and analysed by using GENETYX-MAC version 10.0 (Software Development, Tokyo, Japan). All sequences used for comparison were obtained from GenBank/EMBL/DDBJ. These included (with accession numbers): PTV F65 strain (AJ011380), PEV-9 (Y14459), PEV-8 (AJ001391), poliovirus 1 (PV-1) (V01149), swine vesicular disease virus (SVDV) (X54521), coxsackievirus A 16 (CAV-16) (U05876), human enterovirus 70 (HEV70) (D00820), bovine enterovirus 1 (BEV-1) (D00214), human rhinovirus 14 (HRV-14) (K02121), encephalomyocarditis virus (EMCV) (X74312), mengovirus (Mengo) (L22089), Theiler’s murine encephalomyelitis virus (TMEV) (M20562), foot-and-mouth disease virus A (FMDV-A) (M10975), FMDV-O (X00871), hepatitis A virus (HAV) (M14707), HPeV-1 (L02971), equine rhinitis B virus 2 (ERBV) (X96871) and Aichi virus (Aichi) (AB010145). Phylogenetic trees were constructed by the unweighted pair group method with averages (UPGMA) using GENETYX-MAC version 10.0. Predicted secondary structure formation in the 3'-NTR was analysed by using the program MFOLD (Jaeger et al., 1989 ) with the set of default parameter values in the Wisconsin package version 10.0 (Genetics Computer Group, Madison, WI, USA) and plots were generated by using the program PlotFold (Zuker, 1989 ).
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Results and Discussion |
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TOPAbstractIntroductionMethodsResults and DiscussionReferences |
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). The initiation of translation in the picornavirus genome is considered to be strongly related to the tertiary structure of the internal ribosome entry site (IRES), including a pyrimidine-rich region (Stewart & Semler, 1999 ). A pyrimidine-rich region (UUUCUCU) was located 13 nt prior to the GUG, but not prior to the AUG. It remains to be proven conclusively which codon is used as the initiation codon. Further research on the 5'-NTR will be needed to identify it. The polyprotein coding region was organized as 5'-leader (L) protein–structural proteins–non-structural proteins-3', in which the most obvious difference between the two sequenced PTV genomes and the available sequence of PEV-9 was whether or not they possessed an L protein. The L protein has also been found in cardioviruses, aphthoviruses, ERBV and Aichi virus. However, the Talfan L protein showed no sequence similarity to other picornavirus L proteins or cellular proteins. It remains to be confirmed whether the L protein of Talfan has a protease active site like that of FMDV (Piccone et al., 1995 ). Of all the viral proteins, the amino acid identities to proteins of other picornaviruses were significantly low except for the 2A protein (Table 3). Picornavirus 2A proteins commonly possess protease activity; however, 2A has been reported to be diverse in its structure and function compared with other non-structural proteins. In enteroviruses and rhinoviruses, 2A is a trypsin-like, cysteine protease and is associated with the shut-off of host-cell macromolecular synthesis (Petersen et al., 1999 ), whereas, in cardioviruses, aphthoviruses and ERBV, its processing activity involves an Asn–Pro–Gly–Pro motif (Ryan & Flint, 1997 ) at the 2A/2B junction. Talfan 2A possessed this motif and seemed to be more related to the latter group, indicating that PTVs and PEVs appear to adopt different strategies of multiplication in host cells. This finding might explain the differences in the times that they require for a complete multiplication cycle (data not shown; Morimoto et al., 1962 ). Further genetic studies might account for other idiosyncrasies of these viruses such as physico-chemical properties and different cell culture host ranges.
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; Yamashita et al., 1998 ). All the RdRps of these viruses have been shown to share eight conserved amino acid sequence motifs (Koonin & Dolja, 1993 ). RT–PCR primer sets were constructed covering all eight motifs (Table 2). Consequently, the expected regions of 14 strains were amplified with the Talfan primers, three strains were amplified with the PEV-8/V13 primers and three strains were amplified with the PEV-9/UKG410/73 primers. All the products were sequenced and analysed phylogenetically based on the alignment of the derived amino acid sequences of the concatenated conserved motifs, as described previously (Kaku et al., 1999 ) (Fig. 1). The phylogenetic tree showed that the PEVs were divided into three genetic groups: the Talfan group, the PEV-8/V13 group and the PEV-9/UKG410/73 group. The PEV-9/UKG410/73 group was included in the enterovirus cluster in accordance with current taxonomy, but the PEV-8/V13 group seemed to be less related to it. On the other hand, the remaining 13 strains were located in the same cluster as Talfan, apart from the other PEVs and against the current taxonomy. Interestingly, these genetic groupings were completely consistent with the CPE typing (Fig. 1).
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). The genetic diversity of these protomers seems to be responsible for the idiosyncrasies of picornaviruses such as antigenicity, receptor recognition, buoyant density, pH stability and so on. However, the N terminus of VP2 has been regarded as being in the interior of the mature virion, close to VP4, since VP2 and VP4 are generated from their precursor VP0 by maturation cleavage (VP0
VP2+VP4). Due to its internal location, this region has seemed to be free from the pressure of neutralizing antibodies and to have evolved independently of the neutralizing type. RT–PCR primer sets were constructed covering this region (Table 2). Consequently, the expected regions of 14 strains were amplified with the Talfan primers, three strains were amplified with the PEV-8/V13 primers and two strains were amplified with the PEV-9/UKG410/73 primers. This region of PEV-J10/W47H was not amplified by any primers. All the products were sequenced and approximately 50 derived amino acids at the N terminus of VP2 were aligned by the method of Palmenberg (1989) and then analysed phylogenetically (Fig. 2). The strains were divided into three genetic groups in accordance with CPE typing in the same manner as the RdRp region, giving the Talfan group, PEV-8/V13 group and PEV-9/UKG410/73 group. Within the respective groups, clustering was unlikely to be related to serological classification. This suggested that this region might be more useful for classifying viruses into large clusters such as at the genus level than into antigenicity-related clusters. For detailed study involving the consistent molecular inference of serotypes, it would be essential to analyse the region exposed at the virion surface followed by neutralizing-epitope mapping.
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). The sequences of 3'-RACE products were aligned and compared. Consequently, they were divided into three genetic groups (Fig. 3a). As to length, the 3'-NTR was 62 or 63 nt in the Talfan group, 79 nt in the PEV-8/V13 group and 69 or 72 nt in the PEV-9/UKG410/73 group. The predicted RNA secondary structures varied between groups (Fig. 3b); a single loop was observed in the Talfan group, four loops in the PEV-8/V13 group and two loops in the PEV-9/UKG410/73 group. These folds seemed to be stable, since other folds were not obtained in proximal energy analysis of the sequences of all strains (data not shown). The evolution of the 3'-NTR seems to be closely associated with that of the virus proteins, since this region is recognized by RdRp. These findings will have to be considered along with the efficiency of minus-strand RNA synthesis and the tertiary structure of RdRp of the respective groups in the future.
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Footnotes |
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References |
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TOPAbstractIntroductionMethodsResults and DiscussionReferences |
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Doherty, M., Todd, D., McFerran, N. & Hoey, E. M.(1999). Sequence analysis of a porcine enterovirus serotype 1 isolate: relationships with other picornaviruses. Journal of General Virology 80, 1929-1941.
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Received 10 July 2000;
accepted 20 October 2000.
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