Although echovirus 22 is presently classified as a member of the enterovirus group in the family of picornaviruses, it has been reported to have exceptional biological properties when compared with other representatives of the group. We have determined the complete nucleotide sequence of the echovirus 22 (Harris strain) genome, which appears to be significantly different from all the other studied picornaviruses. However, the organization of the genome [7339 nucleotides, excluding the poly(A) tract] is similar to that of previously sequenced picornaviruses. This genome includes a 5' untranslated region, relatively well-conserved when compared with aphtho-and cardioviruses, followed by an open reading frame coding for a 2180-amino acid-long polyprotein. The amino termini of capsid polypeptides VP1 and VP3 were determined by direct sequencing, and the other proteolytic cleavage sites in the polyprotein were predicted by comparison with other picornavirus proteins. The amino acid identities of echovirus 22 polypeptides with the corresponding proteins of other picornaviruses are in the 14-35% range, similar to those percentages seen when representatives of the five picornavirus groups (entero-, rhino-, cardio-, aphtho-, and hepatoviruses) are compared. Our results suggest that echovirus 22 belongs to an independent group of picornaviruses.

SUMMARYWe have used enzymic amplification of specific nucleic acid sequences followed by hybridization, for the rapid detection and typing of human picornaviruses after cell culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected from the 5' non-coding region of the genome representing highly conserved regions. Sequences specific to enteroviruses and rhinoviruses were used as probes. The assay was able to identify all the picornavirus reference strains analysed and it was also possible to discriminate between enteroviruses and rhinoviruses by the hybridization procedure. When 29 picornavirus clinical isolates were analysed, all except one were detected by gel electrophoresis and a specific hybridization signal was obtained with all except three strains using the oligonucleotide probes. INTRODUCTIONHuman picornaviruses consist of approximately 200 serologically distinct members which are important pathogens. Human rhinoviruses (HRVs) are the most frequent cause of the common cold whereas members of enterovirus subgroups (coxsackie-, echo-and polioviruses) are responsible for e.g. meningitis, paralysis, myositis and myocarditis. Because of the large number of serotypes, specific virological diagnosis of these infections is problematic. Enteroviruses are currently detected using isolation in cell culture followed by neutralization typing with antiserum pools. Rhinoviruses are identified after isolation on the basis of their characteristic acid lability. These methods are laborious and time-consuming because at least two cycles in cell culture are needed for specific recognition of the virus. Since these viruses are common causes of human disease there is an obvious need for new rapid and reliable diagnostic procedures.The nucleotide sequences of approximately a dozen human picornaviruses are already known and these data can be used for the selection of nucleic acid probes for diagnostic purposes. The 5'-terminal non-coding part and regions of the ssRNA genome encoding non-structural proteins are highly conserved among human picornaviruses. Both cloned cDNA (Hyypi~t et al., 1984;Rotbart et al., 1984; A1-Nakib et al., 1986) and synthetic oligonucleotides (Rotbart et al., 1988) have been used successfully as broadly reacting probes in hybridization tests. However, these assays require a passage of the virus in cell culture for amplification of the target. Because it would be desirable to have an assay that could directly demonstrate the presence of virus in clinical samples, we have studied the possibility of using a combination of the polymerase chain reaction (PCR) (Saiki et al., 1988) and oligonucleotide hybridization, for human picornavirus detection. With this aim, we have first considered an assay system and reagents which can be used for identification of picornaviruses after a passage in cell culture. Our results indicate that the system based on these methods can be used for broad detection and typing of ...

The RGD motif is exposed and flexible in common with other known integrin ligands. Although CAV9 resembles coxsackie B viruses (CBVs), several substitutions in the areas implicated in CBV receptor attachment suggest it may recognise a different receptor. The structure along the fivefold axis provides new information on the uncoating mechanism of enteroviruses. CAV9 might bind a larger natural pocket factor than other picornaviruses, an observation of particular relevance to the design of new antiviral compounds.

Detection of enteroviruses and rhinoviruses has traditionally been based on laborious and time-consuming virus isolation. Recently, rapid and sensitive assays for detecting enterovirus and rhinovirus genomic sequences by reverse transcription-polymerase chain reaction (RT-PCR) have been introduced. An RT-PCR assay is described that amplifies both enteroviral and rhinoviral sequences, followed by liquid-phase hybridization carried out in a microtiter plate format. In the hybridization assay, amplicons are identified by enterovirus- or rhinovirus-specific probes carrying lanthanide chelate labels, which can be detected simultaneously by time-resolved fluorometry. The sensitivity and specificity of the RT-PCR-hybridization method were evaluated with a representative collection of enteroviruses and rhinoviruses and tested further its applicability to the clinical setting with cerebrospinal fluid samples and nasopharyngeal aspirates. The RT-PCR assay amplified all enteroviruses and rhinoviruses tested, and all but one amplicon gave a positive result in the subsequent hybridization assay. The RT-PCR-hybridization method was more sensitive than virus isolation for the detection of enteroviruses and rhinoviruses in the clinical samples. High sensitivity, rapidity, and easy performance make the assay suitable for the routine diagnosis of enterovirus and rhinovirus infections.

A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5 untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with genus-specific digoxigenin-labeled oligonucleotide probes. The sensitivity of the multiplex RT-PCR and liquid hybridization assay was 10 to 100 picornavirus genome equivalents for representatives of each picornavirus genus. The hepatitis A virus assay exhibited a sensitivity of 10 genome copies. Both the uniplex and the multiplex tests were highly specific for the target viruses. Twenty-three clinical samples, including cerebrospinal fluid, serum, and nasopharyngeal swab specimens, were used for clinical evaluation of the multiplex RT-PCR assay. The results obtained were consistent with the results of routine virus diagnostic assays. Furthermore, the assay was used to screen 68 stool specimens for the presence of parechoviruses and Aichi virus. One sample was found to contain parechovirus RNA, whereas no Aichi virus was detected. The assay described here can be applied for the efficient identification of human enteroviruses and rhinoviruses in clinical specimens and simultaneously enables the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human parechoviruses and Aichi virus.Picornaviruses include several important human and veterinary pathogens. The large genus of human enteroviruses (HEVs; 64 serotypes) includes polioviruses, coxsackie A and B viruses, echoviruses, and enterovirus types 68 to 71. Although polioviruses will be eradicated in the near future, other HEVs remain a significant cause of morbidity and are responsible for a wide spectrum of clinical symptoms, ranging from mild respiratory infections to severe disease conditions such as myocarditis, meningitis, and encephalitis (6). Human rhinoviruses (HRVs; more than 100 serotypes) are the major cause of the common cold, and they have also been described to have a role in lower respiratory tract infections and exacerbations of asthma (11,16). Despite the availability of vaccines, hepatitis A virus (HAV; a hepatovirus) infections still occur widely. Human parechoviruses (HPEVs), earlier included in the HEVs, currently form their own genus. They appear to cause mainly gastroenteritis and respiratory tract infections, but central nervous system infections have also been associated with these viruses (22). The antibody prevalence of HPEV type 1 (HPEV1) in the Finnish population is greater than 90%, which suggests that the infections may be mild or asymptomatic and underdiagnosed (12). The only known member of the recently established kobuvirus genus, Aichi virus (AV), has been detected in patients wi...