Changing epidemiology of parvovirus B19 in the Netherlands since 1990, including its re-emergence after the COVID-19 pandemic.

Parvovirus B19V (B19V) infection during pregnancy can be complicated by potentially life-threatening fetal hydrops, which can be managed by intrauterine transfusion (IUT). This study investigates the long-term temporal patterns in the epidemiology of B19V and evaluates the impact on fetal hydrops, by combining data on B19V infections from the Dutch Sentinel Surveillance system in the period 1990 to 2023, Dutch blood banking data and hospital data on fetal hydrops. Using wavelet analysis, we identified annual epidemic cycles in the Netherlands in the period 1990-2019 and we identified superimposed multiannual cycles in the period 1990-2009. After 2009, no multiannual cycle could be identified, although the incidence fluctuated and correlates with number of IUT performed. As of 2020, weekly reports of B19V infection demonstrated a historically low incidence and B19V-DNA positive blood donors were nearly absent. From May 2020 to May 2023, no IUT for B19V-related hydrops was performed. In the spring of 2023, B19V infections re-emerged, reaching pre-pandemic epidemic levels. Due to the changes in B19V epidemiology over the last 30 years and the near-absence of B19V during the COVID-19 pandemic, the resulting low immunity levels may lead to rebound outbreaks. Alertness to severe complications such as fetal hydrops is warranted.

In vitro alternative for reactogenicity assessment of outer membrane vesicle based vaccines.

Intrinsic or added immune activating molecules are key for most vaccines to provide desired immunity profiles but may increase systemic reactogenicity. Regulatory agencies require rabbit pyrogen testing (RPT) for demonstration of vaccine reactogenicity. Recently, the monocyte activation test (MAT) gained popularity as in vitro alternative, yet this assay was primarily designed to test pyrogen-free products. The aim was to adjust the MAT to enable testing of pyrogen containing vaccines in an early stage of development where no reference batch is yet available. The MAT and RPT were compared for assessing unknown safety profiles of pertussis outer membrane vesicle (OMV) vaccine candidates to those of Bexsero as surrogate reference vaccine. Pertussis OMVs with wild-type LPS predominantly activated TLR2 and TLR4 and were more reactogenic than Bexsero. However, this reactogenicity profile for pertussis OMVs could be equalized or drastically reduced compared to Bexsero or a whole-cell pertussis vaccine, respectively by dose changing, modifying the LPS, intranasal administration, or a combination of these. Importantly, except for LPS modified products, reactogenicity profiles obtained with the RPT and MAT were comparable. Overall, we demonstrated that this pertussis OMV vaccine candidate has an acceptable safety profile. Furthermore, the MAT proved its applicability to assess reactogenicity levels of pyrogen containing vaccines at multiple stages of vaccine development and could eventually replace rabbit pyrogen testing.

Validation of the monocyte activation test with three therapeutic monoclonal antibodies.

Pharmaceutical products intended for parenteral use must be free from pyrogenic (fever-inducing) contamination. Pyrogens comprise endotoxins from Gram-negative bacteria and non-endotoxin pyrogens from Gram-positive bacteria, viruses, and fungi. The longstanding compendial test for pyrogens is the rabbit pyrogen test, but in 2010 the monocyte acti-vation test (MAT) for pyrogenic and pro-inflammatory contaminants was introduced into the European Pharmacopoeia (Ph. Eur.) as a non-animal replacement for the rabbit pyrogen test. The present study describes the first product-specific Good Manufacturing Practice validation of Ph. Eur. MAT, Quantitative Test, Method A for the testing of three therapeutic monoclonal antibodies. The study used the MAT version with cryo-preserved peripheral blood mononuclear cells and interleukin-6 as the readout. Much of the data presented here for one of the antibodies was included in a successful product license application to the European Medicines Agency.

Parvovirus B19 DNA detectable in hearts of patients with dilated cardiomyopathy, but absent or inactive in blood.

AIMS: Parvovirus B19 (B19V) is often assumed to be a cause of dilated cardiomyopathy (DCM), based on the quantification of B19V DNA in endomyocardial biopsies (EMB). Whether the presence of B19V DNA correlates with active infection is still debated. Application of the enzyme endonuclease to blood samples results in degradation of B19V DNA remnants but leaves viral particles intact, which enables differentiation between active and past infection. In this study, the susceptibility to degradation by endonuclease of B19V DNA in blood was compared between DCM patients and a control group of recent B19V infections. METHODS AND RESULTS: Twenty blood samples from 20 adult patients with DCM, who previously tested positive for B19V DNA in EMB and/or blood, were tested with B19V PCR before and after application of endonuclease to the samples. Six blood samples tested positive for B19V DNA with a mean viral load of 2.3 × 104  IU/mL. In five samples, B19V DNA became undetectable after endonuclease (100% load reduction); in one sample DNA load showed a 23% log load reduction (viral load before endonuclease: 9.1 × 104  IU/mL; after: 6.5 × 103  IU/mL). Presence of cardiac inflammation did not differ between patients with B19V DNAemia (1/4) and patients without B19V DNAemia (6/14) (P value = 1.0). In all 18 control samples of proven recent B19V infections, DNA remained detectable after application of endonuclease, showing only a mean log load reduction of 2.3% (mean viral load before endonuclease: 8.1 × 1011  IU/mL; after: 8.0 × 1011  IU/mL). Load reduction differed significantly between the DCM group and the control group; indicating the presence of intact viral particles in the control group with proven active infection and the presence of DNA remnants in the DCM group (P value = 0.000). CONCLUSION: During recent B19V infection, viral DNA levels in blood were unaffected by endonuclease. In contrast, B19V DNA in blood in patients with DCM became undetectable or strongly reduced after application of endonuclease. Circulating viral DNA in this subset of patients with presumed parvovirus-associated DCM does not consist of intact viral particles. Viral replicative activity cannot be assumed from demonstrating B19V DNA in cardiac tissue or in blood in DCM patients.

Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum.

The monocyte activation test (MAT) is used to detect pyrogens in pharmaceutical products and serves as replacement of the rabbit pyrogen test. The peripheral blood mononuclear cell-based MAT assay requires the addition of serum to the medium and is performed with either fetal bovine serum (FBS) or human serum (HS). Since the capacity to detect non-endotoxin pyrogens (NEPs) in a sensitive manner is an important strength of MAT compared to the bacterial endo­toxin test, the performance of the MAT using FBS and HS was compared using endotoxin and several NEPs. The MAT was more sensitive for endotoxin when FBS was used, however for most NEPs the MAT was more sensitive when per­formed in HS. Furthermore, heat-inactivation of FBS affected the performance of the MAT for endotoxin to some extent but not for the NEPs. Interestingly, heat-inactivation of HS led to an almost complete loss of reactivity towards endotoxin, reduced the response towards heat-killed Staphylococcus aureus and peptidoglycan, but had minor or no effects on the responses towards R848, flagellin, and Pam3CSK4. Product testing of a human blood-derived product in MAT using HS was beneficial since endotoxin spike recoveries were improved. This product is therefore currently batch released with the HS-based MAT assay. Overall, to guarantee optimal performance of MAT, heat-inactivated serum should be avoided. The HS-based MAT appears to be the first choice to replace the rabbit pyrogen test, while in some cases the FBS-based MAT may be favored.

Validation of new real-time polymerase chain reaction assays for detection of hepatitis A virus RNA and parvovirus B19 DNA.

BACKGROUND: To meet European guidelines for plasma for fractionation, plasma fractionators have implemented parvovirus B19 (B19V) and hepatitis A virus (HAV) nucleic acid test (NAT) screening on test pools. In this study we evaluate recently developed in-house NAT assays for B19V DNA and HAV RNA. The B19V NAT was designed to target two different regions of the B19V genome. STUDY DESIGN AND METHODS: The B19V DNA and HAV RNA tests were validated according to commonly used guidelines. The performance of the B19V and HAV assays was evaluated during routine screening of more than 2 × 10(6) donations. RESULTS: The 95% lower limit of detection (LLD) of the HAV NAT was 1.34 IU/mL. The 95% LLD for B19V was 39.1 IU/mL for the NS1 region and 76.9 IU/mL for the VP2 region. The B19V test showed good accuracy, precision, robustness, and no cross-contamination was observed. Both assays detected B19V Genotypes 1 to 3 and HAV Genotypes I to III. During routine screening 103 donations showed B19V DNA loads of more than 1.25 × 10(6) IU/mL and one donation was reactive in the HAV NAT. CONCLUSION: The dual-target B19V polymerase chain reaction (PCR) showed good accuracy (<0.1 log IU/mL) at the crucial concentration of 10 IU/µL for the NS1 and the VP2 region of the B19V genome and detected all known genotypes with similar sensitivity for each genotype. In addition, the dual target format reduces the chance that molecular variants of B19V are wrongly quantified. The HAV RNA assay showed high sensitivity for Genotypes I to III. Both new PCR assays have been successfully introduced for plasma screening in test pools of 480 or 96 donations.

Global co-existence of two evolutionary lineages of parvovirus B19 1a, different in genome-wide synonymous positions.

Parvovirus B19 (B19V) can cause infection in humans. To date, three genotypes of B19V, with subtypes, are known, of which genotype 1a is the most prevalent genotype in the Western world. We sequenced the genome of B19V strains of 65 asymptomatic, recently infected Dutch blood donors, to investigate the spatio-temporal distribution of B19V strains, in the years 2003-2009. The sequences were compared to B19V sequences from Dutch patients with fifth disease, and to global B19V sequences as available from GenBank. All Dutch B19V strains belonged to genotype 1a. Phylogenetic analysis of the strains from Dutch blood donors showed that two groups of genotype 1a co-exist. A clear-cut division into the two groups was also found among the B19V strains from Dutch patients, and among the B19V sequences in GenBank. The two groups of genotype 1a co-exist around the world and do not appear to differ in their ability to cause disease. Strikingly, the two groups of B19V predominantly differ in synonymous mutations, distributed throughout the entire genome of B19V. We propose to call the two groups of B19V genotype 1a respectively subtype 1a1 and 1a2.

Recombinant adeno-associated viral vectors.

Recombinant adeno-associated viral (rAAV) vectors can be used to locally or systemically enhance or silence gene expression. They are relatively nonimmunogenic and can transduce dividing and nondividing cells, and different rAAV serotypes may transduce diverse cell types. Therefore, rAAV vectors are excellent tools to study the function of neuropeptides in local brain areas. In this chapter, we describe a protocol to produce high-titer, in vivo grade, rAAV vector stocks. The protocol includes an Iodixanol gradient, an anion exchange column and a desalting/concentration step and can be used for every serotype. In addition, a short protocol for rAAV injections into the brain and directions on how to detect and localize transduced cells are given.

Both overexpression of agouti-related peptide or neuropeptide Y in the paraventricular nucleus or lateral hypothalamus induce obesity in a neuropeptide- and nucleus specific manner.

Both reduction of melanocortin signaling and increase in neuropeptide Y signaling in the brain result in obesity. However, where in the brain reduced melanocortin or increased neuropeptide Y signaling mediate these effects is poorly understood. In separate experiments we have injected recombinant adeno-associated viral vectors that overexpressed agouti-related peptide or neuropeptide Y in specific brain regions namely the paraventricular nucleus and the lateral hypothalamus. In this review we compare the results from these studies and discuss these data with previous data from intracerebroventricular or local brain injections. This review shows that the effects of agouti-related peptide clearly differ from those of neuropeptide Y. In addition, these data suggests complementary roles for these neuropeptides in energy balance.

Neuropeptide delivery to the brain: a von Willebrand factor signal peptide to direct neuropeptide secretion.

BACKGROUND: Multiple neuropeptides, sometimes with opposing functions, can be produced from one precursor gene. To study the roles of the different neuropeptides encoded by one large precursor we developed a method to overexpress minigenes and establish local secretion. RESULTS: We fused the signal peptide from the Von Willebrand Factor (VWF) to a furin site followed by a processed form of the Agouti related protein (AgRP), AgRP(83-132) or alpha-melanocyte stimulating hormone. In vitro, these minigenes were secreted and biologically active. Additionally, the proteins of the minigenes were not transported into projections of primary neurons, thereby ensuring local release. In vivo administration of VWF-AgRP(83-132), using an adeno-associated viral vector as a delivery vehicle, into the paraventricular hypothalamus increased body weight and food intake of these rats compared to rats which received a control vector. CONCLUSIONS: This study demonstrated that removal of the N-terminal part of full length AgRP and addition of a VWF signal peptide is a successful strategy to deliver neuropeptide minigenes to the brain and establish local neuropeptide secretion.

An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector.

BACKGROUND: This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 x 108 or 1 x 109 genomic copies of AAV1-GFP and 1 x 105 transducing units of LV-dsRed. RESULTS: Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. CONCLUSIONS: This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

Optimization of adeno-associated viral vector-mediated gene delivery to the hypothalamus.

To efficiently deliver genes and short hairpin RNAs to the hypothalamus we aimed to optimize the transduction efficiency of adeno-associated virus (AAV) in the rat hypothalamus. We compared the transduction efficiencies of AAV2 vectors pseudotyped with AAV1, AAV8, and mosaic AAV1/2 and AAV2/8 coats with that of an AAV2 coated vector after injection into the lateral hypothalamus of rats. In addition, we determined the transduction areas and the percentage of neurons infected after injection of various titers and volumes of two AAV1-pseudotyped vectors in the paraventricular hypothalamus (PVN). Successful gene delivery to the hypothalamus was achieved with AAV1-pseudotyped AAV vectors. The optimal approach to transduce an area, with the size of the PVN, was to inject 1 x 10(9) genomic copies of an AAV1-pseudotyped vector in a volume of 1 microl. At a radius of 0.05 mm from the injection site almost all neurons were transduced. In addition, overexpression of AgRP with the optimal approach resulted in an increase in food intake and body weight when compared with AAV-GFP.

Lentivirus-mediated transgene delivery to the hippocampus reveals sub-field specific differences in expression.

BACKGROUND: In the adult hippocampus, the granule cell layer of the dentate gyrus is a heterogeneous structure formed by neurons of different ages, morphologies and electrophysiological properties. Retroviral vectors have been extensively used to transduce cells of the granule cell layer and study their inherent properties in an intact brain environment. In addition, lentivirus-based vectors have been used to deliver transgenes to replicative and non-replicative cells as well, such as post mitotic neurons of the CNS. However, only few studies have been dedicated to address the applicability of these widespread used vectors to hippocampal cells in vivo. Therefore, the aim of this study was to extensively characterize the cell types that are effectively transduced in vivo by VSVg-pseudotyped lentivirus-based vectors in the hippocampus dentate gyrus. RESULTS: In the present study we used Vesicular Stomatitis Virus G glycoprotein-pseudotyped lentivirual vectors to express EGFP from three different promoters in the mouse hippocampus. In contrast to lentiviral transduction of pyramidal cells in CA1, we identified sub-region specific differences in transgene expression in the granule cell layer of the dentate gyrus. Furthermore, we characterized the cell types transduced by these lentiviral vectors, showing that they target primarily neuronal progenitor cells and immature neurons present in the sub-granular zone and more immature layers of the granule cell layer. CONCLUSION: Our observations suggest the existence of intrinsic differences in the permissiveness to lentiviral transduction among various hippocampal cell types. In particular, we show for the first time that mature neurons of the granule cell layer do not express lentivirus-delivered transgenes, despite successful expression in other hippocampal cell types. Therefore, amongst hippocampal granule cells, only adult-generated neurons are target for lentivirus-mediated transgene delivery. These properties make lentiviral vectors excellent systems for overexpression or knockdown of genes in neuronal progenitor cells, immature neurons and adult-generated neurons of the mouse hippocampus in vivo.