Detection of imported wild polioviruses and of vaccine-derived polioviruses by environmental surveillance in Egypt.

Systematic environmental surveillance for poliovirus circulation has been conducted in Egypt since 2000. The surveillance has revealed three independent importations of wild-type poliovirus. In addition, several vaccine-derived polioviruses have been detected in various locations in Egypt. In addition to acute flaccid paralysis (AFP) surveillance, environmental surveillance can be used to monitor the wild poliovirus and vaccine-derived poliovirus circulation in populations in support of polio eradication initiatives.

Isolation of saffold virus type 2 in green monkey kidney cells.

Saffold viruses (SAFV) have been discovered recently and they are classified into Theilovirus species in genus Cardiovirus in the Picornaviridae family. SAFV, especially those belonging to the genotype 2, have been difficult to propagate in laboratory cell lines. This study describes the successful isolation of an efficiently growing SAFV-2 strain directly from a stool specimen by standard virological methods. The availability of SAFV isolates that can be propagated to high titers is crucial to the future studies on pathogenesis and epidemiology of these novel human viruses.

A large Finnish echovirus 30 outbreak was preceded by silent circulation of the same genotype.

An outbreak of echovirus 30 (E-30) in 2009 was confirmed by both frequent isolation of the virus from sewage as well as from patient samples in Finland. Over the last 10 years E-30 had only been isolated sporadically in Finland. We here study the phylogenetic relationships of the strains from the outbreak in the context of E-30 circulation over the last 20 years. The analyzed region comprised 276 nucleotides in the 5' end of VP1 (nucleotides 132-407 in the VP1 of the E-30 Bastianni strain). The Finnish strains were clustered into at least four distinct genogroups, with seven clusters exceeding the genotype demarcation of 12% and the 2009 epidemic strains forming the largest genogroup VII. Moreover, we detected largely divergent genotypes in 2007 and 2009. Interestingly, close genetic relatives of the epidemic strains had already been isolated a few years before the outbreak. Phylodynamic analysis estimated 8.9 years (95% highest posterior density intervals 7.0-11.0) as the age of genogroup VII, indicating a probable origin and evolutionary history prior to its introduction and epidemic expansion in Finland. Finally, the most recent common ancestor for the current E-30 diversity dates back to 1939 (95% highest posterior density intervals 1913-1956).

Molecular epidemiology and dual serotype specificity detection of echovirus 11 strains in Finland.

Echovirus 11 (E-11) has been one of the most frequently discovered human enterovirus (HEV) in Finland during the past few years. We have studied molecular epidemiological patterns of E-11 from 1993 to 2007 exploiting the 257-nucleotide region in the 5'-part of the VP1 used for genetic typing of HEV. Designated genogroup D strains had a striking prevalence among the Finnish strains, a finding in accordance with the recent data from other geographical regions. The subgroup D4, harboring the oldest strains, had become extinct in the beginning of the millennium and D5 strains had taken over. Similarly, a new subgroup of D5 had started to diverge from the main D5 in 2006. However, in addition to endemic D strains, few single strains clustered also to genogroups A and C suggesting importation from more distant locations. The relatively large amino acid sequence variability between and within the genogroups favored the idea of antigenic differences. Neutralization assays confirmed that antigenic differences existed, although all studied E-11 strains were neutralized with antisera against the prototype strain Gregory. Five of the six studied strains belonging to genogroup D were, unexpectedly, also neutralized with antisera against coxsackievirus A9 Griggs.

Eight years of experience with molecular identification of human enteroviruses.

We have successfully typed 1,121 human enterovirus (HEV) isolates during the last 8 years by adapting partial VP1 sequencing to routine identification of HEV isolated from diverse clinical and environmental specimens. The isolates include 48 of the 59 traditional nonpoliovirus HEV serotypes and members of 8 newly discovered types, which would have remained untypeable by neutralization using the conventional cross-sectional pools of antisera.

A whole cell immunization-derived monoclonal antibody that protects cells from coxsackievirus A9 infection binds to both cell surface and virions.

Coxsackievirus A9 (CAV-9) infects human rhabdomyosarcoma (RD) cells using an unidentified RGD-independent receptor. Monoclonal antibodies were prepared by immunizing mice with intact RD cells and by selecting cells from the cytopathic effect of CAV-9 for protection. Here we describe a monoclonal antibody that binds to host cell plasma membrane and protects cells from virus infection. In addition, binding of the virus to cell monolayers was more efficient in the presence of the antibody, suggesting that the antibody is also capable of recognizing virus particles. Immunoprecipitation and electron microscopy studies with highly purified virus preparations verified binding of the monoclonal antibody to the virus particles. The antibody also recognized coxsackievirus A21 and all three serotypes of poliovirus, but without affecting their infectivity. The amino acid sequence of CAV-9 recognized by the monoclonal antibody was identified by peptide mapping and by producing escape mutants in the presence of the antibody.

Characteristics of an environmentally monitored prolonged type 2 vaccine derived poliovirus shedding episode that stopped without intervention.

Vaccine derived poliovirus (VDPV) type 2 strains strongly divergent from the corresponding vaccine strain, Sabin 2, were repeatedly isolated from sewage in Slovakia over a period of 22 months in 2003-2005. Cell cultures of stool specimens from known immune deficient patients and from an identified putative source population of 500 people failed to identify the potential excretor(s) of the virus. The occurrence of VDPV in sewage stopped without any intervention. No paralytic cases were reported in Slovakia during the episode. According to a GenBank search and similarity plotting-analysis, the closest known relative of the first isolate PV2/03/SVK/E783 through all main sections of the genome was the type 2 poliovirus Sabin strain, with nucleotide identities in 5'UTR, P1, P2, P3, and 3'UTR parts of the genome of 88.6, 85.9, 87.3, 88.5, and 94.0 percent, respectively. Phenotypic properties of selected Slovakian aVDPV strains resembled those of VDPV strains isolated from immune deficient individuals with prolonged PV infection (iVDPV), including antigenic changes and moderate neurovirulence in the transgenic mouse model. One hundred and two unique VP1 coding sequences were determined from VDPV strains isolated from 34 sewage specimens. Nucleotide differences from Sabin 2 in the VP1 coding region ranged from 12.5 to 15.6 percent, and reached a maximum of 9.6 percent between the VDPV strains under study. Most of the nucleotide substitutions were synonymous but as many as 93 amino acid positions out of 301 in VP1 showed substitutions. We conclude that (1) individuals with prolonged poliovirus infection are not as rare as suggested by the studies on immune deficient patients known to the health care systems and (2) genetic divergence of VDPV strains may remain extensive during years long replication in humans.

Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland.

BACKGROUND: A nationwide outbreak of hand, foot and mouth disease (HFMD) occurred in Finland in autumn 2008. The outbreak was untypical since a considerable number of clinically diagnosed patients were adults. Furthermore, many of the patients suffered from onychomadesis several weeks after the acute phase of HFMD. OBJECTIVES: Detection, identification and phylogenetic analysis of human enteroviruses (HEV) that caused the outbreak. STUDY DESIGN: A total of 420 clinical specimens were obtained from 317 HFMD cases all over the country. The presence of HEV in the specimens was analysed by virus isolation and/or direct real-time RT-PCR; selected HEV strains were further typed by molecular methods. The genetic similarities of HEV strains were assessed by phylogenetic analyses on partial VP1 sequences. RESULTS: HEV were detected in 212 HFMD cases, including both children and adults, throughout Finland. Two HEV types, coxsackieviruses A6 (CV-A6) and A10 (CV-A10), were identified as the causative agents of the outbreak. One genetic variant of CV-A6 predominated, but, additionally, three other genetically distinct CV-A6 strains were found. All CV-A10 strains segregated into one genetic cluster distinct from previously reported CV-A10 sequences. CONCLUSIONS: The Finnish 2008 HFMD outbreak was caused by two infrequently detected, co-circulating, coxsackie A viruses. Our data suggest endemic circulation of both CV-A types in Northern Europe and that the outbreak was due to the emergence of new genetic variants of these viruses.

Recurrent isolation of poliovirus 3 strains with chimeric capsid protein Vp1 suggests a recombination hot-spot site in Vp1.

Five oral poliovirus vaccine (OPV) strains carrying an intertypic PV3/PV2 recombination in VP1 capsid protein were isolated during poliovirus surveillance. These five PV3 strains had altogether four diverse recombination crossover points near the 3' end of the VP1 coding region. The complete antigenic site IIIa was replaced by PV2-specific amino acids in four of the studied PV3 strains. Low overall number of nucleotide substitutions in VP1 indicated that the predicted replication time, "age", of the PV3 strains was short, 6 months or less. The nucleotide 472-T in the 5' non-coding region, associated to the attenuated phenotype of PV3/Sabin, was reverted to wild-type C in all studied PV3/PV2 recombinant strains. Three of the PV3 strains had at least a tripartite genome deduced from the partial 3D polymerase-coding region sequences. Our results suggest that there exists a PV3/PV2 recombination hot-spot site in the 3' partial region of the VP1 capsid protein and that the recombination may occur within weeks or a few months after the administration of OPV.

Molecular characterization of human rhinovirus field strains isolated during surveillance of enteroviruses.

Human rhinoviruses (HRVs), which are the most frequent causative agents of acute upper respiratory tract infections, are abundant worldwide. We have identified HRV strains in environmental specimens collected in Finland, Latvia and Slovakia during the surveillance of polio- and other enteroviruses. These acid-sensitive HRV strains were isolated under conditions optimized for growth of most of the enteroviruses, i.e. in stationary human rhabdomyosarcoma cells incubated at 36 degrees C. Phylogenetic analysis of the sequences derived from the partial 5' non-coding region and the capsid region coding for proteins VP4/VP2 and VP1 showed that the HRV field strains clustered together with prototype strains of the HRV minor receptor group. Partial sequences of the 3D polymerase coding region generally followed this pattern, with the exception of a set of three HRV field strains that formed a subcluster not close to any of the established HRV-A types, suggesting that recombination may have occurred during evolution of these HRV strains. Phylogenetic analysis of the VP4/VP2 capsid protein coding region showed that the 'environmental' HRV field strains were practically identical to HRV strains recently sequenced by others in Australia, the United States and Japan. Analysis of amino acids corresponding to the intercellular adhesion molecule-1 receptor footprint in major receptor group HRVs and also in the low-density lipoprotein receptor footprint of minor receptor group HRVs showed conservation of the 'minor receptor group-like' amino acids, indicating that the field strains may have maintained their minor receptor group specificity.

An enterovirus strain isolated from diabetic child belongs to a genetic subcluster of echovirus 11, but is also neutralised with monotypic antisera to coxsackievirus A9.

An enterovirus strain (designated D207) isolated from a Slovakian diabetic child and originally serotyped as coxsackievirus A9 (CAV-9) was found to cause rapid cytolysis coinciding with severe functional damage of the surviving cells in primary cultures of human pancreatic islets. This finding prompted us to clone the isolate for full-length genome sequencing and molecular characterization as the prototype strain of CAV-9 is known to cause only minimal damage to insulin-producing beta-cells. Based on capsid-coding sequence comparisons, the isolate turned out to be echovirus 11 (E-11). Phylogenetic analyses demonstrated that E-11/D207 was closely related to a specific subgroup B of E-11 strains known to cause uveitis. To study further antigenic properties of isolate E-11/D207 and uveitis-causing E-11 strains, neutralization experiments were carried out with CAV-9- and E-11-specific antisera. Unlike the prototype strains, the isolate E-11/D207 and uveitis-causing E-11 strains were well neutralized with both CAV-9- and E-11-specific antisera. Attempts to identify recombination of the capsid coding sequences as a reason for double-reactivity using the Simplot analysis failed to reveal major transferred motifs. However, peptide scanning technique was able to identify antigenic regions of capsid proteins of E-11/D207 as well as regions cross-reacting with an antiserum raised to CAV-9. Thus, double specificity of E-11/D207 seems to be a real characteristic shared by the phylogenetically closely related virus strains in the genetic subgroup B of E-11.

Enterovirus surveillance reveals proposed new serotypes and provides new insight into enterovirus 5'-untranslated region evolution.

Human enteroviruses are currently grouped into five species Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. During surveillance for enteroviruses serologically non-typable enterovirus strains were found from acute flaccid paralysis patients and healthy individuals. In this study, we report isolates of recently described enterovirus types EV76 and EV90 of HEV-A species and characterize two new enterovirus type candidates, EV96 and EV97, to species HEV-C and HEV-B, respectively. Analysis of partial 3D regions of EV96 strains revealed sequence divergence consistent with several recombination events between EV96, other HEV-C viruses and polioviruses. Phylogenetic analysis of all available 5'-untranslated region sequences of human entero- and rhinovirus prototype strains and 10 simian enterovirus strains suggested interspecies recombination involving this region.

Enterovirus 94, a proposed new serotype in human enterovirus species D.

The genus Enterovirus (family Picornaviridae) contains five species with strains isolated from humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. In this study, a proposed new serotype of HEV-D was characterized. Four virus strains were isolated from sewage in Egypt and one strain from acute flaccid paralysis cases in the Democratic Republic of the Congo. The complete genome of one environmental isolate, the complete coding sequence of one clinical isolate and complete VP1 regions from the other isolates were sequenced. These isolates had 66.6-69.4% nucleotide similarity and 74.7-76.6% amino acid sequence similarity in the VP1 region with the closest enterovirus serotype, enterovirus 70 (EV70), suggesting that the isolates form a new enterovirus type, tentatively designated enterovirus 94 (EV94). Phylogenetic analyses including sequences of the 5' UTR, VP1 and 3D regions demonstrated that EV94 isolates formed a monophyletic group within the species HEV-D. No evidence of recombination was found between EV94 and the other HEV-D serotypes, EV68 and EV70. Further biological characterization showed that EV94 was acid stable and had a wide cell tropism in vitro. Attempts to prevent replication with protective antibodies to known enterovirus receptors (poliovirus receptor, vitronectin alphavbeta3 receptor and decay accelerating factor) were not successful. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over the past two decades.

Etiology of mumps-like illnesses in children and adolescents vaccinated for measles, mumps, and rubella.

The possible viral etiology of mumps-like illnesses in patients vaccinated for measles, mumps, and rubella (MMR) was studied by use of serum samples prospectively collected, during 1983-1998, from 601 acutely ill Finnish children and adolescents with mumps-like symptoms. Mumps virus was excluded by testing serum samples for mumps antibodies, and the serum samples were further tested for antibodies to adenovirus, enterovirus, Epstein-Barr virus, parainfluenza virus types 1-3, and parvovirus B19. The serum samples of 114 children <4 years old were also tested for antibodies to human herpesvirus 6 (HHV-6). A viral etiology was verified in 84 cases (14%), most commonly Epstein-Barr virus (7%), followed by parainfluenza virus types 1, 2, or 3 (4%) and adenovirus (3%). HHV-6 infection was found in 5 children <4 years old (4%). This study confirms that mumps-like symptoms in MMR-vaccinated children and adolescents are often not caused by mumps virus infection. Careful laboratory-based diagnostic testing of MMR-vaccinated children and adolescents who develop clinical symptoms compatible with those of mumps is important in the treatment of individual patients, in the comprehension of the true epidemiology of these illnesses, and in the evaluation of the impact of MMR vaccination programs.

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child.

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.

Enterovirus infection may induce humoral immune response reacting with islet cell autoantigens in humans.

Molecular mimicry is one of the mechanisms by which enterovirus infections have been postulated to have a role in the pathogenesis of type 1 diabetes. Immunogenic epitopes in enterovirus capsid protein VP1 and procapsid protein VP0 have sequence similarities with diabetes-associated epitopes in tyrosine phosphatase IA-2/IAR and heat shock protein 60. In the present study, documented enterovirus infection was shown to induce humoral responses, that in 7% and 1% of patients cross-reacted with the known diabetes-associated epitopes in tyrosine phosphatase IAR and heat shock protein 60, respectively. In contrast, none of the children vaccinated against poliomyelitis had antibodies to the diabetes-associated epitope of tyrosine phosphatases IA-2/IAR. The antibody response studied in serum samples from six patients with coxsackievirus A9 infection was mainly targeted to capsid protein VP1. Coxsackievirus A9 infection induced antibodies cross-reacted with one epitope in heat shock protein 60, but not with epitopes derived from other autoantigens. Most diabetic children had high levels of antibodies to both coxsackievirus and poliovirus derived VP1 peptides but the pattern of reactivity did not differ from that seen in healthy children. The reactivity of linear epitopes derived from autoantigens was low in general and associated with the presence of multiple autoantibodies in the patients. Some linear auto-epitopes derived from tyrosine phosphatase IA-2, glutamic acid decarboxylase 65, preproinsulin, and heat shock protein 60 were recognized by sera from diabetic patients, but not by sera from healthy children. In conclusion, enteroviruses may induce immune responses that react with islet cell autoantigens, which is a concern when a putative inactivated enterovirus vaccine is considered.