Hein J. Boot

Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype

ABSTRACT Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

Infectious Bursal Disease Virus Capsid Protein VP3 Interacts both with VP1, the RNA-Dependent RNA Polymerase, and with Viral Double-Stranded RNA

ABSTRACT Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus of the Birnaviridae family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we identified the domains responsible for the interaction between VP3 and VP1. Using the yeast two-hybrid system we found that VP1 binds to VP3 through an internal domain, while VP3 interacts with VP1 solely by its carboxy-terminal 10 amino acids. These results were confirmed by using a reverse-genetics system that allowed us to analyze the interaction of carboxy-terminally truncated VP3 molecules with VP1 in infected cells. Coimmunoprecipitations with VP1- and VP3-specific antibodies revealed that the interaction is extremely sensitive to truncation of VP3. The mere deletion of the C-terminal residue reduced coprecipitation almost completely and also fully abolished production of infectious virions. Surprisingly, these experiments additionally revealed that VP3 also binds to RNA. RNase treatments and reverse transcription-PCR analyses of the immunoprecipitates demonstrated that VP3 interacts with dsRNA of both viral genome segments. This interaction is not mediated by the carboxy-terminal domain of VP3 since C-terminal truncations of 1, 5, or 10 residues did not prevent formation of the VP3-dsRNA complexes. VP3 seems to be the key organizer of birnavirus structure, as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1, and the two genomic dsRNAs.

Ability of Lactococcus lactis To Export Viral Capsid Antigens: a Crucial Step for Development of Live Vaccines

ABSTRACT Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.

Assessing the potential effects and cost-effectiveness of programmatic herpes zoster vaccination of elderly in the Netherlands

BackgroundHerpes zoster (HZ) is a painful disease affecting a considerable part of the elderly. Programmatic HZ vaccination of elderly people may considerably reduce HZ morbidity and its related costs, but the extent of these effects is unknown. In this article, the potential effects and cost-effectiveness of programmatic HZ vaccination of elderly in the Netherlands have been assessed according to a framework that was developed to support evidence-based decision making regarding inclusion of new vaccines in the Dutch National Immunization Program.MethodsAn analytical framework was used combining a checklist, which structured relevant data on the vaccine, pathogen and disease, and a cost-effectiveness analysis. The cost-effectiveness analysis was performed from a societal perspective, using a Markov-cohort-model. Simultaneous vaccination with influenza was assumed.ResultsDue to the combination of waning immunity after vaccination and a reduced efficacy of vaccination at high ages, the most optimal cost-effectiveness ratio (€21716 per QALY) for HZ vaccination in the Netherlands was found for 70-year olds. This estimated ratio is just above the socially accepted threshold in the Netherlands of €20000 per QALY. If additional reduction of postherpetic neuralgia was included, the cost-effectiveness ratio improved (~€10000 per QALY) but uncertainty for this scenario is high.ConclusionsVaccination against HZ at the age of 70 years seems marginally cost-effective in the Netherlands. Due to limited vaccine efficacy a considerable part of the disease burden caused by HZ will remain, even with optimal acceptance of programmatic vaccination.

Seroprevalence of seven high-risk HPV types in The Netherlands.

BACKGROUND To obtain insight into the age-specific seroprevalence for seven high-risk human papillomavirus (hr-HPV) serotypes (HPV16, 18, 31, 33, 45, 52, and 58) among the general population in the pre-vaccination era in The Netherlands. METHODS From a cross-sectional population-based study (ISRCTN 20164309) performed in 2006/2007 6384 sera of men, women and children were tested for seven hr-HPV specific antibodies using a fluorescent bead-based multiplex immunoassay with virus-like particles of the seven HPV serotypes. RESULTS An increase in seroprevalence was observed in adolescents, especially for the most prevalent HPV type 16 (up to 11.3%). The increase was most pronounced in women, but was less clear for the other six HPV serotypes. Relatively stable seroprevalences were found in the middle aged cohorts and a slight decrease in the elderly. For the age cohorts >14 years, the seroprevalence among women (25.2%) was higher compared with men (20.3%) (p=0.0002). We found that 10.1% of the population was seropositive for multiple HPV serotypes. CONCLUSIONS The HPV vaccination program is targeted at preadolescents as is justified by the results in this study in which a step-up in HPV seroprevalence is observed at ages of sexual debut. Although direct interpretation of seroprevalence data are hampered by cross-reactivity and seroconversion rate, these data are useful as baseline to evaluate long-term population effects of the HPV16/18 vaccination program.

Generation of full-length cDNA of the two genomic dsRNA segments of Infectious Bursal Disease virus

To determine the complete nucleotide sequence of Infectious Bursal Disease virus (IBDV) isolates, an efficient method was developed to generate full-length cDNA of both the genomic A- and B-segments. Reverse transcription was carried out at the highest possible temperature (50 degrees C) for the reverse transcriptase enzyme, and the single stranded cDNA was subsequently amplified by using an optimized PCR. The double stranded, full-length cDNA was efficiently cloned into a high copy number plasmid. Our results show that the entire cDNA of both the A- and B-segment of a classical attenuated isolate (CEF94), and a very virulent field isolate (D6948), can be cloned. The method will simplify greatly the procedure to generate full-length cDNA and determine the nucleotide sequence of the entire genome of IBDV isolates.

The polio eradication effort has been a great success—let's finish it and replace it with something even better

The polio eradication campaign has greatly reduced the effects of this disease, but many new challenges have emerged. These challenges include the occurrence of polio outbreaks caused by wild-type polioviruses or circulating vaccine-derived polioviruses (cVDPVs) in areas where vaccination coverage is low, the existence of people who excrete poliovirus persistently, and the inability to know definitely that poliovirus has gone. As a result, there is uncertainty about if, when, and how we can end polio immunisation. In this article, we discuss several scenarios for the future of polio control. Because the emergence of cVDPVs necessitates discontinuing the use of live oral polio vaccine, we propose to strive towards a global coverage of near 100% vaccination against all major childhood infections using combination vaccines that contain inactivated poliovirus vaccine. Such a policy will present multiple challenges.

Mumps vaccine effectiveness against orchitis.

To the Editor: Yung et al. reported in the April 2011 issue of Emerging Infectious Diseases on the epidemiologic characteristics of the nationwide mumps outbreak in England and Wales in 2004−2005 (1). The associated effect of disease was considerable, with >43,000 reported cases and >2,600 hospitalizations. Compared with the prevaccine era, the average age of infection was higher, with infection occurring mostly in older teenagers and young adults (2). Older age at infection is associated with a higher risk of certain complications, particularly orchitis (3). Yung et al. reported that among cases of mumps, previous mumps measles rubella (MMR) vaccination offered considerable protection against orchitis, meningitis, and hospitalization (1).

Exchange of the C-Terminal Part of VP3 from Very Virulent Infectious Bursal Disease Virus Results in an Attenuated Virus with a Unique Antigenic Structure

ABSTRACT Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).

Varicella zoster virus infection occurs at a relatively young age in the Netherlands

INTRODUCTION To date, there is no universal varicella vaccination in the Netherlands. We studied the seroprevalence of varicella zoster virus (VZV) specific antibodies and determinants for seropositivity among participants of a serosurveillance study, conducted in 2006/2007 among Dutch inhabitants 0-79 years of age. MATERIALS AND METHODS Serological testing of 6386 blood samples for VZV was performed with a fluorescent bead-based multiplex immunoassay. Seroprevalence and geometric mean concentration (GMC) were weighted for age, sex, ethnicity, and urbanization rate to the total Dutch population. Determinants for VZV seropositivity were identified among children younger than 6 years of age using a logistic regression model. RESULTS The overall seroprevalence of VZV specific antibodies in the Dutch population was 94.6% (95% CI: 93.2-96.0%). This seroprevalence increased rapidly with age: at 6 years of age, more than 95% were seropositive. Determinants associated with lower VZV seropositivity were: young age, first-generation non-Dutch ethnicity, and low frequency of attendance at a day care center or nursery school. The GMC increased with age and was lower for women than for men from the age of 20 years onwards. CONCLUSIONS This study confirmed that VZV infection occurs at a younger age in the Netherlands compared to other countries, which might explain the low disease burden due to varicella. Introduction of universal varicella vaccination is not a foregone conclusion in the Netherlands. Changes in migration and day care usage will influence the age-specific risk on varicella and should therefore be monitored. Further research might elucidate the sex differences in VZV specific GMC.