Enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses

M. Steven Oberste1, Kaija Maher1, David Schnurr2, Mary R. Flemister1, Judith C. Lovchik3, Heather Peters4, Wendy Sessions5, Carol Kirk6, Nando Chatterjee7, Susan Fuller8, J. Michael Hanauer9 and Mark A. Pallansch1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Enterovirus (EV) 68 was originally isolated in California in 1962 from four children with respiratory illness. Since that time, reports of EV68 isolation have been very uncommon. Between 1989 and 2003, 12 additional EV68 clinical isolates were identified and characterized, all of which were obtained from respiratory specimens of patients with respiratory tract illnesses. No EV68 isolates from enteric specimens have been identified from these same laboratories. These recent isolates, as well as the original California strains and human rhinovirus (HRV) 87 (recently shown to be an isolate of EV68 and distinct from the other human rhinoviruses), were compared by partial nucleotide sequencing in three genomic regions (partial sequencing of the 5'-non-translated region and 3D polymerase gene, and complete sequencing of the VP1 capsid gene). The EV68 isolates, including HRV87, were monophyletic in all three regions of the genome. EV68 isolates and HRV87 grew poorly at 37 °C relative to growth at 33 °C and their titres were reduced by incubation at pH 3·0, whereas the control enterovirus, echovirus 11, grew equally well at 33 and 37 °C and its titre was not affected by treatment at pH 3·0. Acid lability and a lower optimum growth temperature are characteristic features of the human rhinoviruses. It is concluded that EV68 is primarily an agent of respiratory disease and that it shares important biological and molecular properties with both the enteroviruses and the rhinoviruses.

The GenBank/EMBL/DDBJ accession numbers reported in this paper are AY426486AY426531.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The human enteroviruses were originally classified on the basis of disease in humans and growth in suckling mice, resulting in four groups: polioviruses, coxsackie A viruses, coxsackie B viruses and echoviruses (Committee on the Enteroviruses, 1957; Pallansch & Roos, 2001). It soon became apparent that some coxsackieviruses were antigenically identical to echoviruses and that some strains of echoviruses were similar to certain coxsackieviruses in causing disease in mice (Committee on the Enteroviruses, 1957, 1962). As a result, the subsequently described enteroviruses were simply called ‘enterovirus' and numbered sequentially, beginning with enterovirus 68 (EV68). Current enterovirus classification takes into account molecular as well as antigenic and biological properties of the viruses, resulting in five human enterovirus species: Poliovirus, Human enterovirus A (HEV-A), HEV-B, HEV-C and HEV-D.

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 2000–2003. 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses.
Seventeen EV68 isolates were available for study, including EV68-Fermon and HRV87-Corn (CA62-1 and CA63, respectively; Table 1). The prototype EV68 strain, EV68-Fermon, was obtained as a research reference reagent from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD, USA); this material is now distributed by the ATCC (VR-1076). The three additional 1962 isolates (Schieble et al., 1967) were obtained from the collection of the Viral and Rickettsial Disease Laboratory, California Department of Health Services (Richmond, CA, USA). The prototype strain of HRV87 was provided by Dean Erdman (Respiratory and Enteric Viruses Branch, CDC, Atlanta, GA, USA). The remaining 12 EV68 strains were isolated in clinical virology laboratories or state public health virology laboratories, from patient specimens obtained between 1989 and 2003 (Table 1). Isolates were obtained by inoculation of clinical specimens into cultures of primary monkey kidney cells and into a variety of continuous cell lines, including A549 (human lung epithelium, CCL-185; ATCC), HLF (human embryonic lung fibroblast; CDC), HFKD (human fetal diploid kidney; Hayflick & Moorhead, 1961), MRC-5 (human embryonic lung fibroblast, CCL-171; ATCC), RD (human rhabdomyosarcoma, CCL-136; ATCC) and WI-38 (human embryonic lung fibroblast, CCL-75; ATCC).


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Table 1. EV68-Fermon prototype strain (CA62-1) and clinical isolates

 
Identification of isolates.
Isolates were typed by neutralization with serotype-specific rabbit antisera or by partial sequencing of the VP1 capsid gene, using primers 292 (5'-MIGCIGYIGARACNGG-3') and 222 (5'-CICCIGGIGGIAYRWACAT-3'), as previously described (Oberste et al., 2000, 2003). Serotype was determined by comparison of the partial VP1 sequence with a database containing complete VP1 sequences for all enterovirus serotypes, as previously described (Oberste et al., 2003).

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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Table 2. Primers for amplification and sequencing of EV68 clinical isolates

 
Sequences for each genome region were aligned with PILEUP (Wisconsin Sequence Analysis Package, version 10.2; Accelrys). The homologous sequences of EV70-J670/71 were included to provide an outgroup for phylogenetic analysis. Pairwise sequence differences were calculated for each genome region using GAP and DISTANCES (Wisconsin Package). Phylogenetic trees were constructed by the neighbour-joining method using the PHYLIP programs DNADIST and NEIGHBOR (version 3.57; http://evolution.genetics.washington.edu/phylip.html), with 1000 bootstrap pseudo-replicates and a transition : transversion ratio of 10. Branch lengths in the consensus tree were calculated using TreePuzzle, version 5.0 (Strimmer & von Haeseler, 1996). Trees were visualized using TreeExplorer, version 2.12 (http://evolgen.biol.metro-u.ac.jp/TE/TE_man.html).

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 ml–1, were calculated by the Kärber formula (Kärber, 1931).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Patients and isolates
The patients from whom the EV68 strains were isolated ranged in age from 7 months to 43 years (Table 1). Thirteen of the 17 patients were 3 years old or younger. The type of illness was specified for 14 patients and in each of these a respiratory illness was noted. Bronchiolitis was the most commonly reported illness, followed by pneumonia. All of the isolates were derived from respiratory specimens, including ten from nasopharyngeal swabs and one from lung tissue (Table 1). Isolates were obtained by inoculation of clinical specimens onto a variety of primary or continuous cell cultures (Table 1). All of the isolates grew in primary monkey kidney cell cultures or in cell lines derived from human lung tissue (Table 1). The 1989–2002 isolates were submitted to the CDC Enterovirus Reference Laboratory for identification because they could not be typed using available antigenic typing reagents; the Texas isolates from 2002 and 2003 were typed by molecular methods by the Texas Department of Health. Partial VP1 sequencing indicated that each of these isolates belonged to the EV68 serotype (data not shown).

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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Table 3. Percentage sequence identity of EV68-Fermon and EV70-J670/71 or members of HEV-A, HEV-B, HEV-C, HRV-A (HRV1B) and HRV-B (HRV14)

 
The EV68-Fermon 5'-NTR nucleotide sequence was 76·1 % identical to that of EV70, 72·5–77·4 % identical to those of HEV-C viruses and less than 71 % identical to those of HEV-A and HEV-B viruses (Table 3). Viruses of species HEV-C and D are known to be closely related to one another in this region of the genome, comprising the enterovirus 5'-NTR cluster I (Hyypiä et al., 1997).

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 64–66 % identical to those of the rhinoviruses in the 5'-NTR, and the amino acid sequences were 48 % identical in P1, 46–51 % identical in P2, 55–57 % identical in P3 and 40–48 % 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. 1b), 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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Fig. 1. Molecular phylogeny of the EV68 prototype strain, EV68-Fermon, based on the nucleotide sequences of the major functional regions of the genome: the 5'-NTR (a), P1 (b), P2 (c) and P3 (d). All trees are plotted to the same scale, as indicated at the bottom of each panel. The positions of representatives of human enterovirus species A–D are indicated. The homologous sequences of human rhinoviruses 1B and 14 were also included. Bar, 0·5 substitutions per site.

 
Clinical isolate sequences
For the clinical isolates, partial genome sequences (partial sequences of the 5'-NTR and partial 3D, and complete VP1 sequences) were generated by standard RT-PCR and sequencing methods using the primers listed in Table 2. The sequences of the clinical isolates were aligned with one another and with the homologous sequences of EV68-Fermon, HRV87-Corn, EV70-J670/71 and enteroviruses of other species. We have previously shown that isolates of a single serotype are generally at least 75 % identical to one another in the complete VP1 sequence (Oberste et al., 1999, 2000). The complete VP1 nucleotide sequences of the clinical isolates were all at least 87 % identical to that of EV68-Fermon and less than 65 % identical to that of any other enterovirus prototype strain (Table 4), confirming their identity as EV68. The partial 5'-NTR sequences of the clinical isolates were 95–100 % identical to one another and 95–99 % identical to that of EV68-Fermon, but only 84–86 % identical to that of EV70 (Table 4). They were no more than 86 % identical to those of other enterovirus serotypes, with viruses of HEV-C being the most closely related, as expected. In the 3D coding region, the clinical isolates were 88–100 % identical to one another in nucleotide sequence, 88–99 % identical to that of EV68-Fermon, 74–79 % identical to that of EV70 and less than 71 % identical to those of other enteroviruses.


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Table 4. Comparison of EV68 clinical isolate nucleotide sequences with each other and with those of EV68-Fermon, HRV87-Corn, EV70-J670/71 and other enteroviruses

 
Temporally and geographically related viruses (the original California isolates, for example) were most closely related to one another in all genome regions, suggesting that they are epidemiologically linked, as proposed during their initial characterization (Schieble et al., 1967). HRV87-Corn also clustered in this group, consistent with its isolation in the same state 1 year later. The most recent isolates, obtained from 1998 to 2003, all clustered together, as did the MN89 and NY93 isolates from 10 years earlier. In contrast to members of other enterovirus species, which are monophyletic by serotype only in the capsid region (Brown et al., 2003; Oberste et al., 2004), all EV68 isolates were monophyletic in all regions of the genome (Fig. 2).



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Fig. 2. Molecular phylogeny of EV68 clinical isolates based on the nucleotide sequences of three genomic regions: the partial 5'-NTR (a), complete VP1 (b) and partial 3D (c). The homologous sequence of EV70-J670/71 was included as the outgroup to orient each tree. All trees are plotted to the same scale, as indicated at the bottom of each panel. Bar, 0·2 substitutions per site.

 
Optimum growth temperature and sensitivity to acid
Two properties that distinguish rhinoviruses from enteroviruses are the instability of rhinoviruses at pH 3 and their lower optimum growth temperature (Hamparian, 1979). It has been reported recently that EV68-Fermon and HRV87-Corn are sensitive to treatment with acid (Blomqvist et al., 2002), despite the original report that EV68-Fermon was acid-resistant (Schieble et al., 1967). To determine whether these two strains were acid-stable and to extend the analysis to include additional isolates, we tested the acid sensitivity of five clinical isolates (MN89, MN98, MD02-1, TX01 and TX02) relative to that of EV68-Fermon, HRV87-Corn, two EV70 strains (J670/71 and USA/FL81-KW43; Hatch et al., 1981) and echovirus 11-Gregory, a representative acid-stable enterovirus (Table 5). The infectivity titres of all of the EV68 strains tested, including EV68-Fermon and HRV87-Corn, were reduced by at least 100-fold after a 1 h incubation in pH 3·0 buffer, relative to incubation in pH 7·2 buffer (Table 5). The titre of EV70-J670/71 was unaltered after acid treatment, but the titre of EV70-USA/FL81-KW43 was reduced more than 1000-fold. The titre of the echovirus 11 control was not affected by acid treatment. Each of the EV68 strains tested also grew to a lower titre at 37 than at 33 °C (Table 5). The differences in titre varied from 10-fold for EV68-Fermon to 1000-fold for strain MN89. The titres of the EV70 strains were approximately equal at the two temperatures, as were the titres of the echovirus 11 control.


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Table 5. Optimum growth temperature and stability in acid of selected EV68 isolates

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The human enteroviruses have long been known to cause respiratory disease (Lennette et al., 1958) and the originally described EV68 isolates were also associated with respiratory illness (Schieble et al., 1967). It is interesting that subsequent EV68 clinical isolates have also originated from respiratory specimens in patients presenting with respiratory symptoms (Table 1) rather than from faecal specimens of aseptic meningitis patients, as is typical for almost all other enteroviruses. HRV87-Corn was originally classified as a rhinovirus based on its association with respiratory disease and its acid lability (Kapikian et al., 1971). Subsequent studies confirmed that the physical properties of HRV87-Corn (virion density and sensitivity to treatment with acid) were similar to those of the rhinoviruses and distinct from those of the enteroviruses (Uncapher et al., 1991). In the original report, EV68-Fermon was described as stable to treatment with acid (Schieble et al., 1967) but it was reported to be acid-labile in a recent comparison with HRV87-Corn (Blomqvist et al., 2002). Our results concur with those of Blomqvist et al. (2002) and demonstrate that additional EV68 clinical isolates are also acid-labile, suggesting that acid lability is a general feature of EV68. This discrepancy with the original EV68 data might be explained by minor differences in technique in the different laboratories, with different cell lines and virus stocks. However, these data also suggest that traditional methods, such as acid lability, may not accurately describe all strains of a given serotype and, in fact, may not always be reproducible for a given strain.

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.


   ACKNOWLEDGEMENTS
 
We are indebted to Jane LaFlash, Wisconsin State Laboratory of Hygiene, and the laboratory staff in other state health departments for their efforts in isolating these viruses. We also thank Dean Erdman, Division of Viral and Rickettsial Diseases, CDC, for providing a stock of HRV87-Corn.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 19 December 2003; accepted 20 May 2004.