1 Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
2 Viral and Rickettsial Disease Laboratory, California Department of Health Services, Richmond, CA, USA
3 Clinical Virology Laboratory, University of Maryland Medical System, Baltimore, MD, USA
4 State of Maryland Department of Health and Mental Hygiene, Baltimore, MD, USA
5 Medical Virology Laboratory, Texas Department of Health, Austin, TX, USA
6 Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, WI, USA
7 Wadsworth Center, New York State Department of Health, Albany, NY, USA
8 Public Health Laboratory, Minnesota Department of Health, Minneapolis, MN, USA
9 Missouri State Public Health Laboratory, Department of Health and Senior Services, Jefferson City, MO, USA
Correspondence
M. Steven Oberste
soberste{at}cdc.gov
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers reported in this paper are AY426486AY426531.
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INTRODUCTION |
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EV68 and EV70 are the only known members of HEV-D. EV70 was first isolated in 1971 as one of two enteroviruses associated with a newly described disease, pandemic acute haemorrhagic conjunctivitis (AHC) (Mirkovic et al., 1973). The other enteroviral agent of AHC is an antigenic variant of coxsackievirus A24, a member of HEV-C (Mirkovic et al., 1974
). EV68 was initially isolated in California in 1962 from four children with pneumonia and bronchiolitis (Schieble et al., 1967
). Since that time, it has been isolated rarely, with only nine isolations reported to the National Enterovirus Surveillance System of the Centers for Disease Control and Prevention (CDC) since its inception in 1970: one in 1987, two in 1994, two in 1997 and once each in the years 20002003. However, antigenic typing reagents are not widely available for EV68, so it is possible that there are EV68 strains among the many untyped enteroviruses isolated in laboratories.
Human rhinovirus 87 (HRV87), which was isolated in 1963 in the same laboratory that isolated the original EV68 strains (Kapikian et al., 1971), is unique among the human rhinoviruses in its receptor usage (Uncapher et al., 1991
). The prototype strain, Corn, is the only known example of HRV87. Recent molecular and antigenic characterization has shown that HRV87-Corn is actually a strain of EV68, based upon cross-neutralization studies and comparison of partial capsid sequences (Blomqvist et al., 2002
; Ishiko et al., 2002
; Savolainen et al., 2002
).
To characterize EV68 further, we have determined the complete genome sequence of the prototype strain, Fermon, and compared it with the previously determined complete sequence of the EV70 prototype strain, J670/71. We have also determined partial genome sequences [partial sequences of the 5'-non-translated (NTR) and 3D polymerase coding regions and the complete VP1 capsid protein coding region sequence] for 15 additional EV68 isolates including the three additional strains that were reported in the original description of EV68-Fermon (Schieble et al., 1967). To our knowledge, this is the first reported characterization of EV68 clinical isolates since the report describing the original 1962 isolates.
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METHODS |
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RT-PCR, sequencing and sequence analysis.
RNA was extracted from infected cell culture supernatants using the QIAamp Viral RNA Mini kit (Qiagen). The complete genomic sequence was determined for EV68-Fermon. Overlapping fragments representing the complete viral genome were amplified by RT-PCR using degenerate, inosine-containing primers designed to anneal to sites encoding amino acid motifs that are highly conserved among enteroviruses, as previously described (Brown et al., 2003; Oberste et al., 2004
). Specific, non-degenerate primers were designed from preliminary sequences to close gaps between the original PCR products. For the other EV68 strains, portions of the 5'-NTR and 3D genes, as well as the complete VP1 gene, were amplified by RT-PCR using standard methods and the primer pairs listed in Table 2
. The PCR products were purified for sequencing using a High-Pure PCR product purification kit (Roche Molecular Biochemicals). For all sequencing, both strands were sequenced by automated methods, using fluorescent dideoxy-chain terminators (Applied Biosystems). The homologous sequences of HRV87-Corn were obtained from GenBank (accession nos AY062273, AY062283 and AY355268).
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Determination of preferred growth temperature and acid lability.
To determine growth efficiency at different temperatures, 10-fold serial dilutions of selected virus strains were inoculated in duplicate onto cells in 96-well cell culture plates. The plates were then incubated at 33 or 37 °C in an atmosphere of 5 % CO2 and examined daily for cytopathic effect for 7 days. For the same selected strains, the sensitivity to treatment with acid was tested by treating the viruses in 0·1 M citrate buffer, pH 3·0, or in 0·1 M phosphate buffer, pH 7·2, in a total volume of 1 ml (Tyrrell & Channock, 1963). Following a 1 h incubation at 37 °C, the virus mixtures were neutralized by the addition of 5 ml 0·5 M phosphate buffer, pH 7·2. Tenfold serial dilutions were prepared in cell culture maintenance medium and inoculated in duplicate onto cells in 96-well cell culture plates. The plates were incubated at 33 °C in an atmosphere of 5 % CO2 and examined daily for cytopathic effect for 7 days. Titres, expressed as TCID50 ml1, were calculated by the Kärber formula (Kärber, 1931
).
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RESULTS |
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Complete genome sequence of EV68-Fermon
To provide a reference point for analysis of the clinical isolates, the complete genome of EV68-Fermon was amplified and sequenced. Overall, the predicted EV68-Fermon polyprotein sequence was 82·5 % identical to that of EV70, the only other known serotype in HEV-D (Table 3). The EV68 and EV70 sequences were most divergent in the capsid region (76·7 % amino acid identity), while the P2 and P3 regions were more highly conserved (84·7 and 87·6 % amino acid identity, respectively) (Table 3
). These similarities are comparable with those observed among heterologous serotypes within other enterovirus species (Brown et al., 2003
; Oberste et al., 2004
). The predicted EV68-Fermon polyprotein sequence was less than 54 % identical to that of viruses of other enterovirus species in the P1 region and less than 62 and 73 % identical in P2 and P3, respectively (Table 3
).
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Because of the association of EV68 with respiratory illness (Table 1), the Fermon sequences were also compared with those of HRV1B and HRV14, representatives of the species Human rhinovirus A and Human rhinovirus B, respectively. The Fermon nucleotide sequences were 6466 % identical to those of the rhinoviruses in the 5'-NTR, and the amino acid sequences were 48 % identical in P1, 4651 % identical in P2, 5557 % identical in P3 and 4048 % identical in the 3'-NTR. Phylogenetic analysis demonstrated that EV68 and EV70 were monophyletic with respect to other enterovirus and rhinovirus species in P1 (Fig. 1
b), P2 (Fig. 1c
) and P3 (Fig. 1d
), as well as in the regions encoding each of the individual mature viral proteins (data not shown). EV68 and EV70 clustered with poliovirus type 1 in the 5'-NTR tree, in agreement with the pairwise comparison data described above (Fig. 1a
).
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DISCUSSION |
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Comparison of the HRV87-Corn sequences with those of the EV68 clinical isolates showed that it clustered closely with the EV68 strains, providing further evidence that HRV87-Corn is a strain of EV68 (Table 4 and Fig. 2
) and in agreement with two recently published analyses (Blomqvist et al., 2002
; Ishiko et al., 2002
). Originally, the relationship between EV68-Fermon and HRV87-Corn was not immediately apparent, despite their isolation in the same laboratory in consecutive years (1962 and 1963). EV68-specific antisera were available but were not used in the initial characterization of HRV87-Corn because Corn was considered a rhinovirus, rather than an enterovirus, due to its acid lability. Thus, it was probably thought that there was no reason to test a rhinovirus with enterovirus reagents, just as such reagents would not have been used to characterize a presumed adenovirus.
Previous studies have shown that recombination occurs frequently among heterotypic enteroviruses of the same species, as demonstrated by incongruent phylogenies for the capsid and non-capsid regions of the genome (Andersson et al., 2002; Brown et al., 2003
; Lukashev et al., 2003
; Oberste et al., 2004
; Santti et al., 1999
, 2000
). In contrast, the EV68 isolates were monophyletic in all genome regions, relative to EV70, providing no evidence for intertypic recombination within HEV-D (Fig. 2
). For recombination to occur, the two parental viruses must be present simultaneously in the same cell. EV68 appears to reside in the respiratory tract. EV70 has been isolated most frequently from conjunctival specimens, but it has occasionally been isolated from extra-ocular sites such as throat swabs and faeces (Nakazono & Kondo, 1989
), suggesting that differences in cell tropism and replication sites may not fully explain the observed lack of recombination between the two serotypes. On the other hand, the current number of isolates of both serotypes within this species that have non-capsid-coding gene sequences available is still quite limited and no other serotype within this species has been described. It remains possible that EV68/EV70 recombinants exist but have not yet been analysed or identified.
The origin of EV70 remains a mystery. It is most closely related genetically to EV68, a potential progenitor whose existence was known at the time of the global emergence of EV70 in the AHC pandemic of 1970; however, the relationship is not so close as to suggest a direct ancestral relationship. It has been hypothesized that EV70 may have emerged through an unknown mechanism from an animal reservoir (Yoshii et al., 1977). One can only speculate that additional members of HEV-D remain to be discovered, either in humans or in animals, and that one of these strains may be closely related to the direct progenitor of the original EV70 AHC strain. It also remains possible that an EV70-related human virus exists, but that it was misidentified as a rhinovirus, analogous to the experience with HRV87-Corn. Molecular characterization of untyped enteroviruses has already shown that additional enterovirus serotypes exist (Norder et al., 2003
; Oberste et al., 2000
, 2001
), suggesting that this strategy may prove fruitful in discovering new members of HEV-D; so far, however, none of the newly discovered serotypes belongs to this species. As new tools become available to screen strain collections rapidly, it will be interesting to see whether any new HEV-D serotypes will emerge.
The exclusive association of EV68 with respiratory disease, the acid lability of EV68 isolates and their poor growth at 37 °C and the apparent mistyping of the HRV87-Corn strain demonstrate that the distinctions between the enteroviruses and the rhinoviruses are neither as clear nor as reliably determined as conventionally believed. As more rhinovirus sequence data become available, the genetic relationships between the enteroviruses and rhinoviruses should be carefully re-examined to determine whether the two genera may be better described as a single genus whose members are polymorphic in some of their properties (e.g. virion density and stability in acid). The currently recognized differences between the enteroviruses and rhinoviruses could then be directly incorporated within the definitions of each species in this combined genus.
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ACKNOWLEDGEMENTS |
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Received 19 December 2003;
accepted 20 May 2004.