؊4 nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.

The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5 noncoding region (5NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5NCR, and the phylogenetic signal in the 5NCR was found to be particularly low. However, some substructure in the 5NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.Within the Picornaviridae family, the Enterovirus genus represents 65 human serotypes which have been classified into five species (A to D and the polioviruses). Members of the human enterovirus B species are the most common cause of aseptic meningitis. They include the six serotypes of group B coxsackieviruses, all echoviruses, coxsackievirus A9, enterovirus 69, and the recently characterized enterovirus 73 (10, 21). Outbreaks of aseptic meningitis typically peak during the summer and early fall, and various serotypes are often associated with a single outbreak (30). The predominant enterovirus types vary from year to year, with echovirus 30, echovirus 13, and echovirus 18 being the most frequently isolated in Europe and the United States over the past few years (2,3,6,7,11,38,41).Laboratory diagnosis of enterovirus infections is currently based on amplification of highly conserved regions within the enteroviral RNA genome (29). The 5Ј noncoding region (5ЈNCR), which contains the cloverleaf and internal ribosome entry site secondary structures, seems to be the most conserved region among enteroviruses and is therefore targeted widely in enterovirus diagnostic procedures (29,38). In addition to traditional ...