Exposure to tick-borne encephalitis virus among nature management workers in the Netherlands.

Tick-borne encephalitis virus (TBEV) has only recently been detected in the Netherlands. With still few autochthonous tick-borne encephalitis (TBE) patients, human exposure to TBEV is expected to be very low among the general population. We aimed to assess the exposure to TBEV among persons with an occupationally high risk of tick bites in the Netherlands. In our cross-sectional serological survey, employees and volunteers of nature management organizations provided a single blood sample and completed an online questionnaire in 2017. The sera were screened in the anti-TBEV IgG Enzyme-Linked Immunosorbent Assay (ELISA), after which a TBEV-specific virus neutralization test (VNT) was applied to confirm positive ELISA outcomes. Ten sera tested positive for IgG antibodies in the TBEV ELISA, among 556 participants who did not report vaccination against TBEV. Through confirmation in VNT, TBEV-specific IgG antibodies were detected among 0.5% (3/556, 95%CI 0.1%-1.6%). During the five years prior to the questionnaire, 87% reported tick bites. Half of the participants considered that most of their tick bites (75% to 100%) had been acquired while being at work. A very low seroprevalence of TBEV exposure was observed among these nature management workers, even though they report a six times higher exposure to tick bites, compared to our general population. Nonetheless, the emergence of TBEV in the Netherlands reaffirms the need for education and preventative measures against tick bites and tick-borne diseases.

The application of an enzyme-linked immunosorbent assay or an immunofluorescent assay test leads to different estimates of seroprevalence of Coxiella burnetii in the population.

The diagnosis and epidemiological studies of Q fever depend on serology. Among the main methods employed are the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescent assay test (IFAT). We show that two commercial assays representing the two methods with two different cut-off titres can lead to significant differences in diagnostic and seroprevalence estimates. This in turn emphasizes the need for a standardized gold method to compare the various assays; whether this standard is 'in-house' or commercially obtained.

Low seroprevalence of Q fever in The Netherlands prior to a series of large outbreaks.

The Netherlands has experienced large community outbreaks of Q fever since 2007. Sera and questionnaires containing epidemiological data from 5654 individuals were obtained in a nationwide seroprevalence survey used to evaluate the National Immunization Programme in 2006-2007. We tested these sera for IgG phase-2 antibodies against Coxiella burnetii with an ELISA to estimate the seroprevalence and to identify determinants for seropositivity before the Q fever outbreaks occurred. Overall seroprevalence was 1·5% [95% confidence interval (CI) 1·3-1·7]. Corrected for confirmation with immunofluorescence results in a subset, the estimated seroprevalence was 2·4%. Seropositivity ranged from 0·48% (95% CI 0·00-0·96) in the 0-4 years age group to 2·30% (95% CI 1·46-3·15) in the 60-79 years age group. Keeping ruminants, increasing age and being born in Turkey were independent risk factors for seropositivity. The low seroprevalence before the start of the outbreaks supports the hypothesis that The Netherlands has been confronted with a newly emerging Q fever problem since spring 2007.

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.

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.

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.

Induction of mucosal immunity by inactivated poliovirus vaccine is dependent on previous mucosal contact with live virus.

The inactivated poliovirus vaccine (IPV) is used for protection against poliomyelitis in The Netherlands. It is not clear, however, whether IPV vaccination can lead to priming of the mucosal immune system and the induction of IgA. It has been demonstrated that IPV vaccination is able to induce strong memory IgA responses in the serum of persons who have been naturally exposed to wild-type poliovirus. This has led to the hypothesis that IPV vaccination is able to induce poliovirus-specific IgA at mucosal sites in persons who have been previously primed with live poliovirus at mucosal sites. To test this hypothesis, the kinetics of the IgA response in serum and saliva after IPV vaccination were examined in persons previously vaccinated with oral poliovirus vaccine (OPV) or IPV. ELISA and enzyme-linked immunospot assays were used for the detection of poliovirus-specific IgA responses. In addition, B cell populations were separated on the basis of the expression of mucosal (alpha4beta7 integrin) and peripheral homing receptors (L-selectin). Parenteral IPV vaccination was able to boost systemic and mucosal IgA responses in previously OPV-vaccinated persons only. None of the previously vaccinated IPV recipients responded with the production of IgA in saliva. In agreement with this finding, a large percentage of the poliovirus-specific IgA-producing lymphocytes detected in previous OPV recipients expressed the alpha4beta7 integrin. It is concluded that IPV vaccination alone is insufficient to induce a mucosal IgA response against poliovirus. In mucosally (OPV-) primed individuals, however, booster vaccination with IPV leads to a strong mucosal IgA response.

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.

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.

Molecular characterization of a wild poliovirus type 3 epidemic in The Netherlands (1992 and 1993).

An outbreak of poliomyelitis due to wild poliovirus type 3 (PV3) occurred in an unvaccinated community in The Netherlands between September 1992 and February 1993. The outbreak involved 71 patients. The aim of this study was to characterize the virus at the molecular level and to analyze the molecular evolution of the epidemic virus. Molecular analysis was carried out by sequencing the VP1/2A junction region (150 nucleotides) of 50 PV3 strains isolated in association with this outbreak and the entire VP1 gene of 14 strains. In addition, the sequence of the VP1/2A junction region of strains from geographical regions endemic for PV3 (Egypt, India, and Central Asia) was analyzed and compared with the nucleotide sequence of the epidemic strain from The Netherlands. The earliest isolate was obtained from river water sampled 3 weeks before diagnosis of the first poliomyelitis patient and was found by VP1/2A sequence analysis to be genetically identical to the strain isolated from the first patient. Sequence divergence among the strains from the epidemic in The Netherlands was less than 2%. The closest genetic similarity (97.3%) was found with an Indian isolate (New Delhi, December 1991), indicating the likely source of the virus. A more than 99% sequence similarity was found in the VP1/2A region. Finally, the sequence information was used to design primers for the specific and highly sensitive molecular detection of PV3 strains during the epidemic.

Isolation of epidemic poliovirus from sewage during the 1992-3 type 3 outbreak in The Netherlands.

To examine the extent of wild poliovirus circulation during the 1992-3 epidemic in the Netherlands caused by poliovirus type 3, 269 samples from sewage pipelines at 120 locations were examined for the presence of poliovirus. The epidemic virus strain was found in 23 samples, all from locations inside the risk area which contained communities that refuse vaccination for religious reasons. By sewage investigation, the wildtype virus was shown to be present in the early phase of the epidemic at two locations, one week before patients were reported from that area. The wild type 3 poliovirus was also detected retrospectively in a river water sample collected for other reasons three weeks before notification of the first poliomyelitis case, at a site a few kilometres upstream the home village of this patient. Oral poliovirus vaccine (OPV) virus was found at 28 locations inside or at the border of the risk area. Trivalent OPV was offered to unvaccinated or incompletely-vaccinated persons living in this region as part of the measures to control the epidemic.

A poliovirus type-specific IgM antibody-capture enzyme-linked immunosorbent assay for the rapid diagnosis of poliomyelitis.

OBJECTIVES: To develop and evaluate an IgM antibody-capture ELISA for the rapid detection of poliovirus type-specific IgM antibodies in serum and CSF of patients suspected of poliomyelitis. STUDY DESIGN: The assay uses monoclonal antibody to human IgM as catching antibody, monovalent IPV as antigen and type-specific monoclonal antibodies labeled with horseradish peroxidase as detecting antibody. RESULTS: Sera from 21 of 24 patients, contacts of these patients or recently vaccinated children were positive in the IgM assay. Sera from 100 healthy individuals were negative in all three poliovirus type-specific ELISAs. Sera from 5 of 81 patients with non-polio viral infections showed (weak) reactivity in one or more of the IgM assays. In a prospective study, sera and/or CSFs from 72 patients with a recent clinical diagnosis of poliomyelitis were tested. A homotypic IgM antibody response was found in 51 of the 55 patients which shedded wild poliovirus in stool or throat. Poliovirus type-specific IgM antibodies were also detected in serum from 13 of the 17 patients, from whom no poliovirus was isolated. Poliovirus type-specific IgM-antibodies could be detected from the second day up till at least 28 days after onset of paralysis. BACKGROUND: Poliovirus infections still create measurable morbidity and mortality in the world. Rapid diagnosis methods are needed to detect infection. CONCLUSION: The use of the poliovirus type-specific IgM antibody-capture ELISA allows rapid diagnosis of poliomyelitis within 24 h after obtaining serum or a CSF specimen.