1 Department of Chemistry and Biomedical Sciences, University of Kalmar, S-391 82 Kalmar, Sweden
2 Nya Apodemus, Grevgatan 38, S-114 53 Stockholm, Sweden
3 University of Texas Medical Branch, Department of Pathology, Centre for Tropical Diseases, Galveston, USA
4 Picornaviral Research Unit, Discipline of Immunology and Microbiology, Faculty of Medical Sciences, The University of Newcastle, David Maddison Clinical Sciences Building, Royal Newcastle Hospital, Newcastle, 2300 New South Wales, Australia
Correspondence
Michael Lindberg
michael.lindberg{at}hik.se
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ABSTRACT |
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The GenBank accession number of the sequence reported in this paper is AF538689.
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MAIN TEXT |
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Three serologically related strains of Ljungan virus (LV) isolated recently from bank voles (Clethrionomys glareolus) are proposed as aetiological agents of myocarditis (Niklasson et al., 1998, 1999
). Sequence analysis of the genomes of three Swedish LV isolates, 87-012 (prototype strain), 174F and 145SL, identified LVs as novel members of the Picornaviridae with several distinctive molecular features (Johansson et al., 2002
). In particular, the LV capsid protein VP1 contains a unique C-terminal extension and the 2A region encompasses a cluster of two diverse 2A homologues. Phylogenetic analysis revealed that the three LV isolates constitute a distinct monophyletic group, which, together with the genus Parechovirus, is separated from other members of the Picornaviridae (Johansson et al., 2002
; Lindberg & Johansson, 2002
).
In the early 1960s during a routine sentinel arbovirus survey in New York, USA, two infectious viruses were isolated from voles (Whitney et al., 1970). Mice inoculated intracerebrally with these strains exhibited either paralysis or convulsions and died within 14 days. Continual attempts to propagate these isolates in cell culture failed. By employing relatively crude biophysical assays, these agents were postulated as putative members of the families Parvo- or Picornaviridae. A third infectious agent, M1146 (Johnson's Microtus montanus enterovirus USA M-1146), isolated in 1962 from voles trapped in Oregon, USA, was shown later to be serologically related to the two New York strains (Johnson, 1965
; Main et al., 1976
). In the present study, the sequence of the M1146 viral genome was determined and analysed for relationships to other members of the Picornaviridae. Molecular characterization confirmed that M1146 is a bona fide member of the Picornaviridae and most closely related to LVs isolated from bank voles in Sweden (Johansson et al., 2002
). On the basis of sequence similarities and predicted genomic organization presented herein, we propose that M1146 should be recognized as a new genotype of LV.
Generation of sufficient quantities of virus for molecular characterization was undertaken initially by intracerebral inoculation of M1146-infected mouse brain suspension and later by two in vitro passages on rhabdomyosarcoma (RD) cells (ATCC), characterized by a mild cytopathic effect. Viral RNA extraction, RT-PCR and nucleotide sequencing strategy were performed as described previously (Johansson et al., 2002). On most occasions, a nested PCR amplification approach was required to obtain sufficient DNA to determine the nucleotide sequence, probably reflecting a low efficiency of virus replication in cell culture. The final genomic sequence was derived from virus propagated in two separate passages in RD cells, extracted and amplified separately to represent the dominant genotype present during propagation in RD cells. Sequences were aligned using the CLUSTAL X program (Thompson et al., 1994
, 1997
) and edited manually using DAMBE [Data Analysis in Molecular Biology and Evolution (Xia, 2000
)]. Prior to phylogenetic analysis, datasets were investigated for the presence of phylogenetic signals corresponding to tree-like evolution using the likelihood mapping method (Strimmer & von Haeseler, 1997
). Phylogenetic reconstruction was conducted by employing the maximum-likelihood method using quartet puzzling, as implemented in TREE-PUZZLE, version 5.0 (Strimmer & von Haeseler, 1996
), based on 1000 puzzling steps. The resulting trees were visualized using the TREEVIEW program (Page, 1996
).
Analysis of the M1146 genome revealed a typical LV-like genomic organization (Fig. 1A). The viral genome is predicted to encode a polyprotein of 2254 aa, followed by a 96 nt long 3'UTR and a poly(A) tail. Despite several attempts with various 5'RACE (rapid amplification of cDNA ends) protocols and amplifications with primers derived from the Swedish LV isolates, only 634 nt of the 3'-proximal region of the M1146 5'UTR was amplified successfully and sequenced. This may be due to a unique 5'UTR sequence of M1146, or to a stable secondary structure of the 5'-terminal region, as predicted previously for the Swedish LVs and human parechoviruses (HPEVs) (Ghazi et al., 1998
; Johansson et al., 2002
). An initiator codon in an optimal Kozak context (Kozak, 1986
) is present at position 635. In the three LV genomes published previously, a second in-frame methionine is encoded 5 aa further downstream from the putative initiating methionine residue (Fig. 1C
) (Johansson et al., 2002
). A corresponding alternate initiator codon is not present in the M1146 genome, supporting previous predictions concerning the location of the start codons in the Swedish LV genomes.
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Phylogenetic relationships of the 3Dpol protein sequences with LVs and representative members of the Picornaviridae revealed a close relationship between M1146 and the Swedish LVs (Fig. 2A). All LVs (M1146, 87-012, 174F and 145SL) cluster together in a distinct monophyletic group that is more related to the genus Parechovirus than to other picornaviruses. Analysis of the relationship between LVs and HPEVs using the capsid protein precursor (P1) showed further that, although LVs and HPEVs are related, these clades are clearly distinct from each other (Fig. 2B
). Furthermore, this analysis indicated that the four LVs characterized to date cluster geographically and by vole species, with M1146 being more distant to the closely related Swedish LV isolates.
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The 3'UTRs of the Swedish LVs have been predicted previously to fold into two hairpins, structurally similar to those identified in the 3'UTR of poliovirus (Auvinen & Hyypiä, 1990; Johansson et al., 2002
; Pöyry et al., 1996
). M1146 possesses a shorter 3'UTR (96 nt) than the Swedish LVs (107111 nt). The predicted RNA secondary structure of M1146 3'UTR [MFOLD 3.1 (Zuker et al., 1999
) and STAR (Abrahams et al., 1990
) programs] was shown to vary compared to the stemloops in the Swedish LVs (Fig. 3
B, C). Interestingly, both the primary sequence of the 3' one-third of the 3'UTR and the predicted stemloop structure thereof (domain II) are highly conserved among all four LVs (Fig. 3
). In contrast, the primary sequence of the 5'-proximal two-thirds of M1146 3'UTR is only distantly related to the corresponding region of the 3'UTR of the Swedish LVs, with no specific affinity found with any picornavirus or sequence of other origin (data not shown). This region of M1146 3'UTR is predicted to fold into a less stable and more open stemloop structure that differs considerably to the more stable domain I predicted for the closely related Swedish LVs. The functional significance of these different predicted structures on the LV replication should be revealed in future studies.
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In agreement with this finding, recent analysis of the protein composition of LV 87-012 by SDS-PAGE revealed a slightly larger molecular mass of VP1 than if the VP1|2A1 site was processed as predicted previously by sequence analysis (E. S. Johansson, D. R. Shafren, G. Frisk, T. Hyypiä, K. Edman & A. M. Lindberg, unpublished results). However, the observed molecular mass corresponds to the size of a potential VP12A1 fusion protein. Hence, the DvExNPG|P motif may be included at the C terminus of VP1. Aphthovirus 2A-like sequences encoding this motif have not only been identified in several members of the Picornaviridae but also at various locations in picorna-like insect viruses (Govan et al., 2000; Isawa et al., 1998
; Johnson & Christian, 1998
; Pringle et al., 1999
; Wilson et al., 2000
; Wu et al., 2002
). In some of these viruses, the aphthovirus 2A-like sequence appears to mediate polyprotein processing both between individual capsid proteins and between the structural precursor and the non-structural protein region (Donnelly et al., 2001a
; Wu et al., 2002
). Taken together, these data imply that the VP1|2A1 border proposed previously may not be processed in any of the LV genomes. Consequently, the aphthovirus 2A-like homologue in the LV polyproteins may be the C terminus of capsid protein VP1 and mediate polyprotein processing in a manner similar to that suggested for the picorna-like insect viruses.
In conclusion, we have employed molecular characterization to confirm that the virus, M1146, isolated from voles in the early 1960s in the USA indeed is a member of the Picornaviridae. Despite being isolated on two continents from different rodent species spanning more than 30 years, molecular comparison revealed that M1146 is most closely related to LVs isolated from bank voles in Sweden (Johansson et al., 2002). Identification of LVs in both Swedish and North American voles suggests a continued presence of LVs over a wide geographical range throughout numerous vole populations. The M1146 non-structural proteins exhibited a high degree of similarity to the Swedish LV proteins, while the deduced protein sequence of the P1 region was more divergent. Therefore, M1146 represents a new genotype of the LV clade, prototyped by LV 87-012. Data presented herein together with distinct molecular features of LV reported previously (Johansson et al., 2002
) strongly support the establishment of a new genus of picornavirus.
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ACKNOWLEDGEMENTS |
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Received 23 August 2002;
accepted 12 December 2002.