Alignment of capsid protein VP1 sequences of all human rhinovirus prototype strains: conserved motifs and functional domains.

An alignment was made of the deduced amino acid sequences of the entire capsid protein VP1 of all human rhinovirus (HRV) prototype strains to examine conserved motifs in the primary structure. A set of previously proposed crucially important amino acids in the footprints of the two known receptor molecules was not conserved in a receptor group-specific way. In contrast, VP1 and VP3 amino acids in the minor receptor-group strains corresponding to most of the predicted ICAM-1 footprint definitely differed from those of the ICAM-1-using major receptor-group strains. Previous antiviral-sensitivity classification showed an almost-complete agreement with the species classification and a fair correlation with amino acids aligning in the antiviral pocket. It was concluded that systematic alignment of sequences of related virus strains can be used to test hypotheses derived from molecular studies of individual model viruses and to generate ideas for future studies on virus structure and replication.

Environmental surveillance of wild poliovirus circulation in Egypt--balancing between detection sensitivity and workload.

Examination of sewage specimens for poliovirus (environmental surveillance) was adopted as a supplementary tool in the surveillance of poliomyelitis in Egypt. Sewage samples were concentrated about 50-fold using a simple two-phase separation technique, and inoculated in cell cultures in two collaborating laboratories in parallel. All but 9 of the 293 (97%) samples collected from January 2001 to December 2002 contained poliovirus and/or other enteroviruses, with polioviruses being detected in 84% of the samples. The proportion of specimens containing type 1 wild poliovirus (PV1W, the North-East African (NEAF) genotype) was less in 2002 (16%) than in 2001 (57%), and further decreased in 2003. While the overall sensitivity to detect PV1W was similar in the two collaborating laboratories, the specimens scored positive were not identical. Parallel cultures inoculated with aliquots of a given specimen very frequently resulted in isolation of different viruses. Moreover, partial sequence analysis occasionally revealed representatives of different genetic lineages of PV1W in a given specimen. These results emphasize the need to use intensive laboratory analysis to optimise sample sensitivity in environmental poliovirus surveillance, and the difficulties in reproducing the isolation results by simple re-inoculation of samples containing a mixture of different viruses.

Phylogenetic analysis of human rhinovirus capsid protein VP1 and 2A protease coding sequences confirms shared genus-like relationships with human enteroviruses.

Phylogenetic analysis of the capsid protein VP1 coding sequences of all 101 human rhinovirus (HRV) prototype strains revealed two major genetic clusters, similar to that of the previously reported VP4/VP2 coding sequences, representing the established two species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Pairwise nucleotide identities varied from 61 to 98 % within and from 46 to 55 % between the two HRV species. Interserotypic sequence identities in both HRV species were more variable than those within any Human enterovirus (HEV) species in the same family. This means that unequivocal serotype identification by VP1 sequence analysis used for HEV strains may not always be possible for HRV isolates. On the other hand, a comprehensive insight into the relationships between VP1 and partial 2A sequences of HRV and HEV revealed a genus-like situation. Distribution of pairwise nucleotide identity values between these genera varied from 41 to 54 % in the VP1 coding region, similar to those between heterologous members of the two HRV species. Alignment of the deduced amino acid sequences revealed more fully conserved amino acid residues between HRV-B and polioviruses than between the two HRV species. In phylogenetic trees, where all HRVs and representatives from all HEV species were included, the two HRV species did not cluster together but behaved like members of the same genus as the HEVs. In conclusion, from a phylogenetic point of view, there are no good reasons to keep these two human picornavirus genera taxonomically separated.

Sequence analysis of human rhinoviruses in the RNA-dependent RNA polymerase coding region reveals large within-species variation.

Human rhinoviruses (HRVs; family Picornaviridae), the most frequent causative agents of respiratory infections, comprise more than 100 distinct serotypes. According to previous phylogenetic analysis of the VP4/VP2-coding sequences, all but one of the HRV prototype strains distribute between the two established species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Here, partial sequences of the RNA-dependent RNA polymerase (3D polymerase)-coding gene of 48 HRV prototype strains and 12 field isolates were analysed. The designated division of the HRV strains into the species HRV-A and HRV-B was also seen in the 3D-coding region. Phylogenetically, HRV-B clustered closer to human enterovirus (HEV) species HEV-B, HEV-C and poliovirus than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D-coding region than in the VP4/VP2-coding region, with the difference maxima reaching 48 % at the nucleotide level and 36 % at the amino acid level in HRV-A and 53 and 35 %, respectively, in HRV-B. Within both species, a few strains formed a separate cluster differing from the majority of strains as much as HEV-B from HEV-C. Furthermore, the tree topology within HRV-A differed from that for VP4/VP2, suggesting possible recombination events in the evolutionary history of the strains. However, all 12 field isolates clustered similarly, as in the capsid region. These results showed that the within-species variation in the 3D region is greater in HRV than in HEV. Furthermore, HRV variation in the 3D region exceeds that in the capsid-coding region.

Characterization of a highly evolved vaccine-derived poliovirus type 3 isolated from sewage in Estonia.

Two types of vaccine-derived polioviruses have been recently designated to emphasize the different origins of the evolved viruses: circulating vaccine-derived polioviruses (cVDPV) associated with outbreaks of paralytic disease and strains isolated from chronically infected immunodeficient individuals (iVDPV). We describe here a type 3 VDPV (PV3/EST/02/E252; later E252) isolated from sewage collected in Tallinn, Estonia, in October 2002. Due to aberrant properties in subtyping, the virus was subjected to detailed characterization. Partial genomic sequencing suggested that the closest relative was the oral vaccine strain PV3/Sabin, but the two virus strains shared only 86.7% of the 900 nucleotides (nt) coding for the capsid protein VP1. Phylogenetic analysis of the nearly complete genome [nt 19 to poly(A)] revealed multiple nucleotide substitutions throughout the genome and a possible Sabin 3/Sabin 1-recombination junction site in the 2C coding region. A calculation based on the estimated mutation frequency of the P1 region of polioviruses suggested that the E252 virus might have replicated in one or more individuals for approximately 10 years. No persons chronically excreting poliovirus are known in Estonia. Amino acid substitutions were seen in all known antigenic sites, which was consistent with the observed aberrant antigenic properties of the virus demonstrated by both monoclonal antibodies and human sera from vaccinated children. In spite of the apparent transmission potential, no evidence was obtained for circulation of the virus in the Estonian population.

Evolution of wild-type 1 poliovirus in two healthy siblings excreting the virus over a period of 6 months.

Wild-type 1 poliovirus (wtPV1) strains were isolated from two young healthy brothers shortly after arrival in Finland from Somalia in 1993. Twelve (sibling A) and 18 (sibling B) specimens collected over a period of more than 6 months yielded wtPV1. Partial sequences obtained from the one and two earliest isolates from sibling A and B, respectively, were nearly identical, differing from each other by only one or two nucleotides. Subsequently, the virus evolved separately in both siblings so that maximal differences between strains derived from a given subject peaked at 2.2 % for sibling A, at 1.5 % for sibling B and at 2.5 % between the two siblings in the VP1-coding part of the genome. All substitutions in the 150 nt VP1-2A junction region were synonymous, whereas as many as eight of the 31 variable positions in the remaining VP1-coding region encoded amino acid replacements in at least one strain. Probable structural locations of the variable amino acid positions were mapped to the published PV1/Mahoney structural model. Most of the substitutions occurred around the fivefold axis in motifs that are known to be or suspected to be targets of neutralizing antibodies. We suggest that the striking genetic divergence observed between the strains was based on a combination of bottleneck transmission events and antigenic drift during the prolonged period of poliovirus replication.

Human rhinoviruses.

Human rhinoviruses are the most important causative agents of upper respiratory infections and are also implicated in more severe clinical entities. Although often present, very little is known about human rhinoviruses. Molecular methods have been used in the classification of this large group of viruses into two separate clades. In addition, one known serotype was found to be a member of enterovirus group D. Laboratory diagnosis of human rhinovirus infection is based on reverse transcription polymerase chain reaction methods or the more tedious virus culture but a rapid "bedside" method is unavailable. Anti-rhinoviral therapy has been under extensive study over the past few decades but symptomatic treatment of the common cold is still the only useful approach in clinical use. More data on circulating human rhinovirus strains would facilitate both detection and treatment of these common pathogens.

Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species.

Enteroviruses show a high degree of sequence variation both between and within serotypes due to the lack of proofreading of the viral RNA-dependent RNA polymerase. In addition, recombination is known to occur not only within but also between different serotypes. We have previously shown that capsid coding sequences of coxsackievirus B4 (CVB4) cluster in several coexisting genotypes (intergenotypic nucleotide difference of 12 % or more) whereas a single lineage of echovirus 30 (EV30) has been prevailing and evolving throughout the last two decades. In the major capsid gene, VP1, clustering of both nucleotide and amino acid sequences correlates with serotype. We have now determined a 501 nucleotide sequence in the non-structural 3CD region of CVB4 and EV30 field strains. Phylogenetic analysis revealed that sequences of Human enterovirus B (HEV-B) were segregated in the 3CD region into three distinct clusters without the VP1-associated serotype/genotype correlation. One of the clusters comprised the E2 strain of CVB4, the EV30 prototype and five other CVB4 field strains whereas the other two clusters, in addition to CVB4 and EV30 strains, also included other HEV-B serotypes. We believe that intertypic recombination is the most likely explanation for the observed incongruence. Similarity analysis based on complete genomes of the CVB4 and EV30 prototypes and the CVB4 E2 strain revealed that a putative recombination spot was mapped within the 2B gene. The incongruence observed in the two genomic domains (P1 and P3) suggests a certain degree of independent evolution, which may be explained by interserotypic recombination within an enterovirus species. It is thus difficult to exclude recombination in the history of any given strain.

Human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features.

It has recently been reported that all but one of the 102 known serotypes of the genus Rhinovirus segregate into two genetic clusters (C. Savolainen, S. Blomqvist, M. N. Mulders, and T. Hovi, J. Gen. Virol. 83:333-340, 2002). The only exception is human rhinovirus 87 (HRV87). Here we demonstrate that HRV87 is genetically and antigenically highly similar to enterovirus 68 (EV68) and is related to EV70, the other member of human enterovirus group D. The partial nucleotide sequences of the 5' untranslated region, capsid regions VP4/VP2 and VP1, and the 3D RNA polymerase gene of the HRV87 prototype strain F02-3607 Corn showed 97.3, 97.8, 95.2, and 95.9% identity to the corresponding regions of EV68 prototype strain Fermon. The amino acid identities were 100 and 98.1% for the products of the two capsid regions and 97.9% for 3D RNA polymerase. Antigenic cross-reaction between HRV87 and EV68 was indicated by microneutralization with monotypic antisera. Phylogenetic analysis showed definite clustering of HRV87 and EV68 with EV70 for all sequences examined. Both HRV87 and EV68 were shown to be acid sensitive by two different assays, while EV70 was acid resistant, which is typical of enteroviruses. The cytopathic effect induced by HRV87 or EV68 was inhibited by monoclonal antibodies to the decay-accelerating factor known to be the receptor of EV70. We conclude that HRV87 and EV68 are strains of the same picornavirus serotype presenting features of both rhinoviruses and enteroviruses.

Phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons.

Human rhinoviruses (HRV) have been shown to be the major causative agent for mild respiratory infections, but also associated with more serious diseases, such as acute otitis media and pneumonia in children, and asthma. Despite the economical and medical importance of HRV, little is known about the circulation and genetic diversity of HRV during a given season. The aim of this study was to genetically characterize HRV strains causing acute respiratory infections in a cohort of small children during a 2 years follow-up time. Genetic relationships between 61 HRV field isolates were studied using partial genomic sequencing in the VP4/VP2 region (420 nt) and phylogenetic analysis of these sequences. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades, the designated genetic group 2 (including HRV 14) being more predominant. The maximum genetic variation within group 1 was 32.3% and within group 2 it was 32.7%. Several distinct clusters could be observed, some of which were strictly seasonal, whereas some other variants were detected during several seasons. The results of this study show striking genetic diversity of the HRV strains circulating in a given community during a short time.

Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70.

Human rhinoviruses (HRV), common agents of respiratory infections, comprise 102 designated serotypes. The genetic relationships of HRV prototype strains and the possibility of using genetic identification of a given HRV field strain were studied. Genomic sequences in the VP4/VP2 region were obtained from all 102 prototype strains. Phylogenetic analysis included 61 recently isolated Finnish field strains. Seventy-six out of the 102 prototype strains clustered in the HRV genetic group A and 25 in group B. Serotype 87 clustered separately and together with human enterovirus 70. The 'percentage' interserotypic differences were generally similar to those between different enterovirus serotypes, but for six pairs of HRV serotypes they were less than 10%. The maximum variation in genetic group A was 41% at the nucleotide level and 28% at the amino acid level, and in genetic group B 34% and 20%, respectively. Judging from the observed interserotypic differences, the 61 Finnish field isolates might represent as many as 19 different serotypes. One cluster of the field strains did not directly associate with any of the prototype strains and might represent a new serotype. However, larger numbers of field isolates of known serotype need to be characterized, possibly also in the VP1 region, to evaluate the feasibility of genetic typing of HRV strains.