Continued seasonal circulation of enterovirus D68 in the Netherlands, 2011-2014.

Enterovirus D68 (EV-D68) continued to circulate in a seasonal pattern in the Netherlands, after the outbreak in 2010. Outpatient EV-D68 cases, mainly in the under 20 and 50­59 years age groups, presented with relatively mild respiratory disease. Hospital-based enterovirus surveillance identified more severe cases, mainly in children under 10 years of age. Dutch partial VP1 genomic region sequences from 2012 through 2014 were distributed over three sublineages similar to EV-D68 from the outbreak in the US in 2014.

Immunity against poliomyelitis in the Netherlands, assessed in 2006 to 2007: the importance of completing a vaccination series.

Europe has been declared polio-free since 2002. Here we describe the seroprotection against poliomyelitis in the Dutch population using banked serum samples. Samples from 1,581 inhabitants of eight municipalities with low vaccination coverage (LVC) and an additional 6,386 samples from a nationwide (NS) group (clinical trial number: ISRCTN20164309; collected in 2006­07) were tested for neutralising antibodies (log² reciprocal titres (GMT); non-protection <3) against all three poliomyelitis serotypes. Demographic and epidemiological data were used for statistical regression analysis. Seroprevalence in the NS was 94.6% (type 1), 91.8% (type 2) and 84.0% (type 3). Infants (0­7 months-old) had ≥80% seroprevalence for all serotypes. The highest seroprevalence was found in children, with type 1 and type 2 in five year-olds and type 3 in nine to 10 year-olds. In the LVC group, orthodox protestants, many of whom refuse vaccination, showed seroprevalence rates of 64.9% (type 1), 61.0% (type 2) and 62.1% (type 3). In the NS group, non-Western immigrants and travellers to non-European continents had higher seroprevalences compared to Western immigrants and travellers within Europe, respectively. The Dutch National Immunisation Programme against poliomyelitis has provided good seroprotection, with high and long-lasting GMTs against all serotypes upon completion. The unvaccinated population remains at risk.

Detection of human enteroviruses and parechoviruses as part of the national enterovirus surveillance in the Netherlands, 1996-2011.

Laboratories of the Dutch Working Group on Clinical Virology have routinely performed enterovirus diagnostics in the Netherlands since the early 1960s, with country-wide coverage. Enterovirus-positive samples are typed for clinical and epidemiological purposes, as well as to document the absence of poliovirus circulation. Human parechoviruses 1 and 2, initially recognized as enteroviruses, and since 2006 also the higher numbered human parechovirus types, have been detected as part of this surveillance. The purpose of this report is to describe the national enterovirus surveillance data from stool specimens collected in the Netherlands between 1996 and 2011 by all the participating laboratories. Since 2007, the average annual percentage of human enterovirus- and parechovirus-positive specimens increased from 6.5 to 10.8% and from 0.3 to 2.5% of the total numbers of specimens tested, respectively, following a gradual implementation of molecular diagnostics directly on clinical samples. Increased detection rates were observed for human enterovirus species A coxsackieviruses (from 0.1 to 0.5%). Human enteroviruses of species B, C, and D were detected at average rates of 4.7, 0.04, and 0.005%, respectively. The introduction of molecular diagnostics also resulted in an increase in the number of untyped enterovirus-positive specimens for which the presence of poliovirus was not excluded (from 1.3 to 3.1% since 2007). To increase knowledge on human entero- and parechovirus epidemiology and type-specific pathogenesis, as well as to warrant the quality of the poliovirus surveillance in the Netherlands, it is of importance to continue the typing of enterovirus- and parechovirus-positive samples.

[Poliomyelitis in 2006: all or nothing]. / Poliomyelitis in 2006: alles of niets.

Since it was launched in 1988, the Global Polio Eradication Initiative has made great progress: millions of children have been saved from a serious disease and type 2 poliovirus has been eradicated. The original target of polio eradication by the year 2000 was, however, too optimistic: as of 2006, polio remains endemic in 4 countries (Nigeria, India, Pakistan and Afghanistan). In northern Nigeria, it is particularly difficult to reach enough children during National Immunisation Days to stop the circulation of wild poliovirus and prevent spread to neighbouring countries that are at risk for epidemics due to low routine vaccination coverage. However, critics of the initiative provide no real alternative. The WHO will continue the initiative using new strategies to realise--for the second time in history after smallpox--the eradication ofa serious infectious disease.

Poliovirus circulation among schoolchildren during the early phase of the 1992-1993 poliomyelitis outbreak in The Netherlands.

During the 1992-1993 outbreak of poliomyelitis in The Netherlands, we examined 866 childrenat 7 schools for evidence of infection with the outbreak virus, poliovirus type 3(PV3), to determine the extent of the outbreak and the protection of the herd immunity. Seventy-seven children (8.9%) showed evidence of recent wild-type PV3 infection, as determined by virus isolation and/or poliovirus type-specific IgM assay. Most infected children lived in the same area as the index case patient, attended an orthodox-reformed (OR) primary school, and had not been vaccinated. At the OR school, as many as 22% of children immunized with inactive poliovirus vaccine were found to have evidence of recent infection, which is a significantly lower rate than that among unvaccinated children (59.5%). No evidence of vaccination was seen in 25.5%-43.1% of children at OR schools. Seroprevalence of antibodies against the 3 types of poliovirus suggested that no poliovirus circulation had occurred between the 1978 and 1992-1993 outbreaks.

Antibody responses to antigenic sites 1 and 3 of serotype 3 poliovirus after vaccination with oral live attenuated or inactivated poliovirus vaccine and after natural exposure.

Three important antigenic sites involved in virus neutralization on polioviruses in mouse experiments have been identified. These sites are located at the surface of the virion and have been designated antigenic sites 1, 2, and 3. In mice, the antibody response to antigenic site 1 of serotype 3 poliovirus is considered to be immunodominant, but little is known about the immunogenicity of these sites in humans. In the present study, we developed inhibition enzyme-linked immunosorbent assays specific for antigenic sites 1 and 3 to measure antibody responses to these sites in fully vaccinated inactivated poliovirus vaccine (IPV) (n = 63) and oral live attenuated poliovirus vaccine (OPV) (n = 63) recipients and in naturally infected persons (n = 25). Similar levels of antibodies to site 1 in IPV and OPV vaccinees were detected. However, significantly more OPV recipients (88.7%) had detectable antibodies to antigenic site 3 (P < 0.01) than did IPV-vaccinated persons (63. 1%). After an IPV booster vaccination, both previously IPV- and OPV-vaccinated persons responded with a significant increase in antibodies to sites 1 and 3 (P < 0.01). We conclude that the immune response to serotype 3 poliovirus in humans consists of both site 1- and site 3-specific antibodies and that these responses can be induced by either OPV or recent IPV vaccination.

Lessons from diagnostic investigations of patients with poliomyelitis and their direct contacts for the present surveillance of acute flaccid paralysis.

One of the key strategies for the global eradication of poliomyelitis is the virological investigation of stool samples in cases of acute flaccid paralysis (AFP) to exclude poliovirus as a possible cause. Clinical specimens from a serotype 3 outbreak provided an opportunity to examine the potential of newly developed methods for the diagnosis of poliomyelitis. The virus isolation rate was maximal (89.6%) during the first 2 weeks after the onset of paralysis and then dropped sharply to 18.6%. In contrast, a high percentage of patients tested positive for poliovirus-specific IgM (93.9%) in the early phase of the infection and remained positive for up to 8 weeks. Virus isolation would have correctly identified only 54.9% of the AFP cases. This rate would have been increased to 92% through the use of the poliovirus-specific IgM ELISA. The IgM ELISA could serve as an important additional tool for the rapid diagnosis of poliomyelitis.

Genetic basis for immunological aberrations in poliovirus Sabin serotype 3 strains imported in the netherlands.

During the characterization of poliovirus type 3 strains imported in The Netherlands, Sabin serotype 3 strains that reacted with both specific antisera against Sabin-like (vaccine) and non-Sabin-like (wild-type) strains by the intratypic strain differentiation assay have been found. The present study was done to determine the pathogenic potential of these virus strains for humans. Characterization of these so-called double-reactive strains with neutralizing monoclonal antibodies (MAbs) against the major antigenic sites of serotype 3 Sabin virus led to the identification of two groups with different antigenic properties. Six of the seven strains were resistant to neutralization with MAbs against sites 2B and 3B and one strain was neutralized by all the MAbs in a manner similar to that for the Sabin serotype 3 virus. Partial sequencing of the coding regions confirmed the antigenic changes for all six antigenically distinct strains. By inoculation of these viruses into transgenic mice which express the human poliovirus receptor, one strain was identified as highly neurovirulent, three were identified as intermediate, and three were identified as attenuated. Sera from vaccinated persons efficiently neutralized the mutants. Our data suggest that some double-reactive strains are a potential risk to the unvaccinated community but not to the vaccinated population.

Molecular epidemiology of poliovirus infection in Tunisia.

This report is an overview of poliomyelitis surveillance in Tunisia from 1991 to 1996. In all, 2088 stool specimens, collected from 152 acute flaccid paralysis (AFP) cases and from 1747 of their healthy contacts were investigated. Virus isolation was done systematically in RD and HEp-2C cell lines and isolated viruses were typed by sero-neutralisation as polioviruses or non-polio enteroviruses. Poliovirus isolates were analysed systematically for their wild or vaccine-related origin by two methods--one based on antigenic differences and one on genetic differences between strains. All type 2 polioviruses were vaccine-related and most wild viruses belonged to polio serotype 3. Wild polio type 3 viruses were detected in 1991 and 1992 in six cases of paralytic polio. A silent circulation of wild polio 1 and wild polio 3 was detected in 1994. No wild virus was detected in Tunisia from 1995 onwards. Wild polioviruses were sequenced and compared with Tunisian wild strains isolated during the 1980s, as well as other genotypes from the international database. These investigations revealed a single Tunisian polio 3 genotype that has been circulating from 1985 to 1994 and two different polio 1 genotypes. These results reflect effective control strategies within the country and contribute to the improvement of the polio eradication programme effectiveness at national and global levels.

Australia's last reported case of wild poliovirus infection.

In 1986 tests on faeces collected from a 22 year old Australian born man who had symptoms consistent with poliomyelitis yielded poliovirus type 3. In a neutralisation test using a panel of monoclonal antibodies the isolate was identified as wild poliovirus type 3 at that time. After further classification using microneutralisation, nucleic acid probe hybridisation, immunoassay and sequencing carried out in three laboratories between 1994 and 1997, the isolate has been reclassified as 'Sabin-like' with 'wild type' characteristics. This case has been quoted in the literature as Australia's last case of locally acquired wild poliovirus. Efforts are now being made to identify the true last case in Australia. This article describes the isolation, identification and further characterisation of this virus.

A Sabin vaccine-derived field isolate of poliovirus type 1 displaying aberrant phenotypic and genetic features, including a deletion in antigenic site 1.

Poliovirus strains derived from the oral poliovirus vaccine (Sabin) can be differentiated from wild-type poliovirus by tests based on either immunological or genetic properties of the strains. The characterization of a recently identified poliovirus type 1 isolate with exceptional properties is described. Initial phenotypic analysis of the virus by use of polyclonal absorbed antisera suggested a wild-type character. However, the different genomic analyses all confirmed the Sabin-derived character of the virus. All 17 plaques isolated from the strain shared these properties, thus excluding the possibility of a mixture of a wild-type and a Sabin-derived strain. To elucidate the properties of this virus further, the nucleotide sequences of the P1 region and most of the 5' non-coding region were established. Although the nucleotide identity with Sabin 1 was more than 99.4%, mutations were observed in regions encoding three major antigenic sites; the deduced amino acid substitutions confirmed the aberrant results of micro-neutralization assays with site-specific monoclonal antibodies. The most striking feature was the existence of a hexanucleotide deletion in the VP1 gene, which gave rise to a two amino acid deletion in the BC loop. In spite of these antigenic changes, the strain was readily serotyped as poliovirus type 1 under standard conditions. Likewise, replication of the virus under cell culture conditions was not affected by these mutations or by the deletion. Standard polio vaccination protects against this aberrant virus, and its epidemiological significance remains open.

Multicenter evaluation of the Amplicor Enterovirus PCR test with cerebrospinal fluid from patients with aseptic meningitis. The European Union Concerted Action on Viral Meningitis and Encephalitis.

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the "gold standard" were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.

Antigenic and molecular characterization of wild type 1 poliovirus causing outbreaks of poliomyelitis in Albania and neighboring countries in 1996.

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus.

Genetic analysis of wild-type poliovirus importation into The Netherlands (1979-1995).

Wild polioviruses were isolated a number of times in The Netherlands outside the epidemic periods (1978 and 1992-1993) from patients infected abroad, from subclinically infected persons, and from river water. Sequence comparisons revealed discrete sources of importation: the Mediterranean, India, and Indonesia. The observed wide genetic variation is indicative of repeated importation and not of indigenous circulation. Isolates identical or closely related to the epidemic type 1 strain of 1978 were found in clinical and environmental specimens until 1983, probably due to repeated importation from Turkey. Viruses related to the 1992-1993 epidemic type 3 virus had already been isolated six times before the epidemic. Of particular importance are two documented isolations of prototype wild poliovirus indistinguishable from that used to produce the inactivated vaccine. These data underscore the continued risk to the unvaccinated religious population of exposure to wild poliovirus.

Poliovirus-specific immunoglobulin A in persons vaccinated with inactivated poliovirus vaccine in The Netherlands.

In The Netherlands the inactivated poliovirus vaccine (IPV) is used for protection against poliomyelitis. It is not clear if parenteral vaccination with IPV can lead to priming of the mucosal immune system. We developed and evaluated enzyme-linked immunosorbent assays for the detection of poliovirus serotype-specific immunoglobulin A (IgA) and secretory IgA antibodies. Using these assays we examined the kinetics of the IgA response in sequential serum samples from 15 poliomyelitis patients after natural infection with serotype 3 poliovirus. In 36% of the patients IgA remained present for up to 5 months postinfection. Furthermore, we examined, in an IPV-vaccinated population, the presence of IgA antibodies in sera from young children (4 to 12 years of age; n = 177), sera from older children (between 13 and 15 years of age; n = 123), sera from healthy blood donors (n = 66), and sera from naturally immune elderly persons (n = 54). The seroprevalence of IgA to all three serotypes was low in young vaccinated children (5 to 7%), and the seroprevalence of IgA types 2 and 3 was low in older vaccinated children (2 to 3%). The seroprevalence of antibodies to type 1 was significantly higher (18%) in older children than in younger children. This higher seroprevalence is most likely explained by the persistence of IgA following infection with the serotype 1 wild-type poliovirus strain during the 1978 epidemic. In healthy adults, the seroprevalence of type 1- and type 2-specific IgA was significantly higher than that in young children. These results suggest that at least part of the IgA found in the older population is induced by infections unrelated to the IPV vaccination schedule. Finally, we found that parenteral vaccination with IPV was able to boost secretory IgA responses in 74 to 87% of a naturally exposed elderly population (n = 54). While the presence of secretory IgA in IPV-vaccinated persons has been documented previously, our findings suggest that mucosal priming with live virus is necessary to obtain an IgA response after IPV booster vaccination.

['Open season in the hunt for poliovirus' (continued)]. / 'De jacht op het poliovirus is geopend' (vervolg).

Global eradication of poliomyelitis was started in 1988. Polio eradication is considered feasible on theoretical and practical grounds, is cost-effective and is endorsed at the highest political levels. The strategy is based on (a) increasing vaccination coverage through routine immunization, national immunization days and so-called mopping-up campaigns, (b) improving surveillance, and (c) polio-free certification. Since the start of the programme global vaccination coverage increased from 67% in 1988 to 83% in 1995; the number of reported cases--an estimated 10% of the total number--decreased by 90% from 35,251 (1988) to 3,755 (1996). A rapid further decrease is expected with the start of national immunization days on the Indian subcontinent. In the next years the emphasis will be strongly on surveillance i.e. detection of possible polio patients and (wild) poliovirus circulation. All countries will need to implement reliable surveillance to show their polio-free status. Only then can the world be declared polio-free.

The molecular epidemiology of type 1 poliovirus in Central African Republic.

An increase in the incidence of acute flaccid paralysis cases associated with wild-type 1 poliovirus occurred in children in the city of Bangui, Central African Republic (CAR), in 1993 and 1994. Genetic relationships of 33 isolates were analysed by restriction fragment length polymorphism and by sequencing the VP1/2A junction region (150 nucleotides) of the viral genome. Two distinct genotypes, A and B, were co-circulating in 1993, while in 1994 only a third genotype, C, was observed. Comparison of the sequences found, with those of the sequences from isolates from neighbouring and other endemic countries revealed that genotype A isolates were related to strains from Egypt (90.7% identity), genotype B isolates to strains from Kenya (96.7% identity), Sudan and Egypt, and genotype C isolates to strains from various countries in western and southern Africa (89.0% identity). Genotypic diversity and genetic linkage with strains from neighbouring countries indicate intense poliovirus circulation and transmission that does not respect national borders. Therefore, eradication of poliomyelitis from CAR can only be achieved by a coordinated multinational strategy that stops poliovirus circulation in the whole of Africa.

Evaluation of a poliovirus-binding inhibition assay as an alternative to the virus neutralization test.

An enzyme-linked immunosorbent assay (ELISA)-based poliovirus-binding inhibition (PoBI) test to detect and quantify antibodies to polioviruses was optimized and evaluated for use in population studies as an alternative to the virus neutralization test (NT) in tissue culture. The sensitivities of the inhibition ELISA compared with the NT in an inactivated poliovirus vaccine (IPV)-vaccinated population were 98.6, 97.4, and 92.1% for serotypes 1, 2, and 3, respectively. The specificities of the PoBI test, as determined with sera from nonvaccinated persons, were also high for all three serotypes (99.0, 95.8, and 100%, respectively). Antibodies to other enteroviruses did not cross-react in the serotype 1 and 3 PoBI, and only levels of cross-reactivity were found for serotype 2. We found high correlations between the PoBI and NT titers for serotypes 1 and 2 in IPV-vaccinated blood donors (0.97 and 0.95), in oral poliovirus vaccine (OPV)-vaccinated blood donors (0.91 and 0.95), and in naturally immune persons (0.90 and 0.87). The correlation coefficient for serotype 3, however, was significantly lower in OPV-vaccinated blood donors (0.73) and in naturally immune persons (0.76) than in IPV-vaccinated persons (0.94; P < 0.01). These results indicate that the antibody response to serotype 3 poliovirus in IPV recipients is different from that in OPV recipients and naturally infected persons. We conclude that the PoBI test is a suitable alternative to the NT for estimating the seroprevalence of neutralizing antibodies to poliovirus, especially in large-scale population studies.