Subjective health and psychosomatic complaints of children and adolescents in Germany: Results of the HBSC study 2009/10 - 2022.

Background: Subjective health and well-being are important health indicators in childhood and adolescence. This article shows current results and trends over time between 2009/10 and 2022. Methods: The Health Behaviour in School-aged Children (HBSC) study examined subjective health, life satisfaction and psychosomatic complaints of N = 21,788 students aged 11 to 15 years in the school years 2009/10, 2013/14, 2017/18 and in the calendar year 2022. Multivariate regression analyses show the associations between sociodemographic characteristics and well-being in 2022, as well as trends since 2009/10. Results: The majority of children and adolescents indicate a good subjective health and high life satisfaction. About half of the girls and one third of the boys report multiple psychosomatic health complaints, with a clear increase over time. Older adolescents, girls and gender diverse adolescents are at an increased risk of poor well-being. Subjective health and life satisfaction varied between 2009/10 and 2022, with a significant deterioration between 2017/18 and 2022. Conclusions: The high proportion of children and adolescents with psychosomatic complaints, as well as the observed gender and age differences, underline the need for target group-specific prevention, health promotion and continuous health monitoring.

[Epidemiology of mental well-being in childhood and adolescence. Results from three epidemiological studies before and during the COVID-19 pandemic]. / Epidemiologie seelischen Wohlbefindens von Kindern und Jugendlichen in Deutschland. Ergebnisse aus 3 Studien vor und während der COVID-19-Pandemie.

BACKGROUND: Continuous nationwide health monitoring is important to track the well-being of children and adolescents and to map developmental trajectories. Based on the results of three selected epidemiological studies, developments in child well-being over the past 20 years are presented. METHODS: Data are based on (1) the mental health module of the German National Health Interview and Examination Survey among Children and Adolescents (BELLA study, 2003-2017, N = 1500 to 3000), which is a module of the KiGGS study; (2) the COvid-19 and PSYchological Health Study (COPSY, 2020-2022, N = 1600-1700), which is based on the BELLA Study; and (3) the International Health-Behaviour in School-aged Children Study (HBSC, 2002-2018, N = 4300-7300). Well-being was assessed in 7­ to 17-year-olds using indicators of health-related quality of life (KIDSCREEN-10), life satisfaction (Cantril Ladder), and mental health problems (Strenghts and Difficulties Questionnaire (SDQ), Screen for Child Anxiety Related Emotional Disorders (SCARED), and Center for Epidemiological Studies Depression Scale for Children (CES-DC)). RESULTS: Overall, children and adolescents show consistently high health-related quality of life and high overall life satisfaction pre-pandemic (2002-2018), which initially worsened with the onset of the 2020 COVID-19-pandemic. Two years later, improvements are evident but have not yet reached baseline levels. Psychological problems, as well as symptoms of anxiety and depression, increased by up to 12 percentage points at the beginning of the pandemic and are still higher two years after the onset of the pandemic compared to pre-pandemic studies. CONCLUSION: The epidemiology of child well-being provides a necessary data basis to assess the support needs of children and adolescents and to use this as a basis for developing measures of health promotion, prevention, and intervention.

Horizontal transmission of endemic viruses among rhesus macaques (Macaca mulatta): Implications for human cytomegalovirus vaccine/challenge design.

INTRODUCTION: Rhesus macaques are natural hosts to multiple viruses including rhesus cytomegalovirus (RhCMV), rhesus rhadinovirus (RRV), and Simian Foamy Virus (SFV). While viral infections are ubiquitous, viral transmissions to uninfected animals are incompletely defined. Management procedures of macaque colonies include cohorts that are Specific Pathogen Free (SPF). Greater understanding of viral transmission would augment SPF protocols. Moreover, vaccine/challenge studies of human viruses would be enhanced by leveraging transmission of macaque viruses to recapitulate expected challenges of human vaccine trials. MATERIALS AND METHODS: This study characterizes viral transmissions to uninfected animals following inadvertent introduction of RhCMV/RRV/SFV-infected adults to a cohort of uninfected juveniles. Following co-housing with virus-positive adults, juveniles were serially evaluated for viral infection. RESULTS: Horizontal viral transmission was rapid and absolute, reaching 100% penetrance between 19 and 78 weeks. CONCLUSIONS: This study provides insights into viral natural histories with implications for colony management and modeling vaccine-mediated immune protection studies.

Robust validation and performance comparison of immunogenicity assays assessing IgG and neutralizing antibodies to SARS-CoV-2.

To enable benchmarking of immunogenicity between candidate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there is a need for standardized, validated immunogenicity assays. In this article, we report the design and criteria used to validate immunogenicity assays and the outcome of the validation of serologic and functional assays for the evaluation of functional immune response and antibody titers in human serum. A quantitative cell-based microneutralization (MNT) assay, utilizing a reference standard, for detecting anti-SARS-CoV-2 spike protein-neutralizing antibodies in human serum and Meso Scale Discovery's multiplex electrochemiluminescence (MSD ECL) assay for immunoglobulin G (IgG) antibodies to SARS-CoV-2 spike, nucleocapsid, and receptor-binding domain (RBD) proteins were assessed for precision, accuracy, dilutional linearity, selectivity, and specificity using pooled human serum from coronavirus disease 2019 (COVID-19)-confirmed recovered donors. Both assays met prespecified acceptance criteria for precision, relative accuracy, dilutional linearity, selectivity, and specificity. Both assays demonstrated high specificity for the different SARS-CoV-2 antigens or virus tested, and no significant cross-reactivity with seasonal coronaviruses. An evaluation to compare the neutralizing activity in the MNT assay to the IgG measured using the MSD ECL assay showed a strong correlation between the presence of neutralizing activity and amount of antibodies against the spike and RBD proteins in sera from both convalescent and vaccinated individuals. Finally, the MNT assay was calibrated to the WHO reference standard to enable reporting of results in international units, thus facilitating comparison of immunogenicity data generated by different assays and/or laboratories. The MSD ECL assay has previously been calibrated. In conclusion, these validated assays for the evaluation of functional immune response and antibody titers following SARS-CoV-2 vaccination could provide a relatively simple standardized approach for accurately comparing immune responses to different vaccines and/or vaccination regimens.

Toward the Development of a Sustainable Scientific Research Culture in Azerbaijan (2011-2015).

This review especially describes the dangerous pathogens research program in Azerbaijan (AJ) funded by the US Defense Threat Reduction Agency under the Cooperative Biological Engagement Program (CBEP) from 2011 through 2015. The objectives of the CBEP are to prevent the proliferation of biological weapons; to consolidate and secure collections of dangerous pathogens in central repositories; to strengthen biosafety and biosecurity of laboratory facilities; and to improve partner nations' ability to detect, diagnose, report, and respond to outbreaks of disease caused by especially dangerous pathogens. One of the missions of the CBEP is therefore to increase the research skills and proficiency of partner country scientists. The program aims to fulfill this mission by sponsoring scientific research projects that exercise the modern diagnostic techniques available in the CBEP-engaged laboratories and the enhanced disease surveillance/control programs. To strengthen the local scientists' ability to develop research ideas, write grant proposals, and conduct research independently, in-country CBEP integrating contractor personnel have mentored scientists across AJ and conducted workshops to address technical gaps. As a result of CBEP engagement, seven research projects developed and led by AJ scientists have been funded, and five projects are currently in various stages of implementation. The Defense Threat Reduction Agency has also sponsored AJ scientist participation at international scientific conferences to introduce and integrate them into the global scientific community. The efforts summarized in this review represent the first steps in an ongoing process that will ultimately provide AJ scientists with the skills and resources to plan and implement research projects of local and regional relevance.

Implementation and evaluation of a training program as part of the Cooperative Biological Engagement Program in Azerbaijan.

A training program for animal and human health professionals has been implemented in Azerbaijan through a joint agreement between the United States Defense Threat Reduction Agency and the Government of Azerbaijan. The training program is administered as part of the Cooperative Biological Engagement Program, and targets key employees in Azerbaijan's disease surveillance system including physicians, veterinarians, epidemiologists, and laboratory personnel. Training is aimed at improving detection, diagnosis, and response to especially dangerous pathogens (EDPs), although the techniques and methodologies can be applied to other pathogens and diseases of concern. Biosafety and biosecurity training is provided to all trainees within the program. Prior to 2014, a variety of international agencies and organizations provided training, which resulted in gaps related to lack of coordination of training materials and content. In 2014 a new training program was implemented in order to address those gaps. This paper provides an overview of the Cooperative Biological Engagement Program training program in Azerbaijan, a description of how the program fits into existing national training infrastructure, and an evaluation of the new program's effectiveness to date. Long-term sustainability of the program is also discussed.

Limited dissemination and shedding of the UL128 complex-intact, UL/b'-defective rhesus cytomegalovirus strain 180.92.

UNLABELLED: The UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b' region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b' region predicts acute endothelial tropism and long-term shedding in vivo in the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b' region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropism in vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b' region, rapidly emerged during in vivo replication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuated in vivo and suggest that additional UL/b' genes, besides the UL128 complex, are required for optimal in vivo CMV replication and dissemination. IMPORTANCE: An effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate the in vivo function of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance of in vivo studies in evaluating human CMV gene function and suggest that additional UL/b' genes are required for optimal CMV dissemination and transmission.

Use of specific-pathogen-free (SPF) rhesus macaques to better model oral pediatric cytomegalovirus infection.

BACKGROUND: Congenital human cytomegalovirus (HCMV) infection can result in lifelong neurological deficits. Seronegative pregnant woman often acquire primary HCMV from clinically asymptomatic, but HCMV-shedding children. METHODS: Potential age-related differences in viral and immune parameters of primary RhCMV infection were examined in an oral rhesus CMV infection model in specific pathogen free macaques. RhCMV shedding was measured by real time PCR in plasma, saliva and urine. Immune parameters, including neutralizing and binding antibodies and RhCMV-specific T cell responses, were assessed in longitudinally collected blood samples. RESULTS: The oral RhCMV infection model in infant SPF rhesus macaques demonstrated that (i) the susceptibility to oral RhCMV infection declines with age, and (ii) infant macaques shed RhCMV more persistently and at higher titers compared to adult macaques. (iii) CONCLUSIONS: The oral infant RhCMV infection model appears to reflect viral pathogenesis in human HCMV-infected children. Larger studies are needed to define immune parameters associated with better control of RhCMV in adult compared to young animals.

Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys.

Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b' region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b' region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b' region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one naïve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques.

The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.

Analysis of rhesus rhadinovirus microRNAs expressed in virus-induced tumors from infected rhesus macaques.

Rhesus rhadinovirus (RRV), a primate gamma-herpesvirus related to human Kaposi's sarcoma-associated herpesvirus (KSHV), causes a similar pattern of pathogenesis. Previously, RRV was shown to express 7 pre-microRNAs (pre-miRNAs) in latently infected cells. Using deep sequencing, we analyzed the pattern of small RNA expression in vivo using latently RRV-infected B-cell lymphoma and retroperitoneal fibromatosis tissues. We identified 15 virally encoded pre-miRNAs in both tumors, including all previously reported RRV pre-miRNAs. Although all 15 RRV pre-miRNAs, like all 12 KSHV pre-miRNAs, are located 3' to the conserved viral ORF71 gene and in the same transcriptional orientation, only one RRV miRNA is homologous to a KSHV miRNA. One previously identified RRV miRNA, miR-rR1-3, is actually a miRNA offset RNA (moRNA) derived from sequences located adjacent to pre-miR-rR1-3. Several other RRV-derived moRNAs were obtained, including one recovered >600 times. Together, this research provides a comprehensive list of the miRNAs and moRNAs encoded by RRV.

A heterologous DNA prime/protein boost immunization strategy for rhesus cytomegalovirus.

A previous study in nonhuman primates demonstrated that genetic immunization against the rhesus cytomegalovirus phosphoprotein 65-2 (pp65-2) and glycoprotein B (gB) antigens both stimulated antigen-specific antibodies and CD8 T cell responses, and significantly reduced plasma viral loads following intravenous challenge with RhCMV. It was also noted in this study that weak CD4 T cell and neutralizing antibody responses were generated by DNA alone. To broaden the type of immune responses, a DNA prime/protein boost strategy was used in seronegative macaques, consisting of four DNA immunizations against pp65-2, gB, and immediate-early 1 (IE1), followed by two boosts with formalin-inactivated RhCMV virions. This heterologous prime/boost strategy elicited robust antigen-specific CD4 and CD8 T cell responses in addition to biologically relevant neutralizing antibody titers. Animals were challenged with RhCMV delivered into four sites via a subcutaneous route. Skin biopsies of one of the inoculation sites 7 days post challenge revealed marked differences in the level of RhCMV replication between the vaccinated and control monkeys. Whereas the inoculation site in the controls was noted for a prominent inflammatory response and numerous cytomegalic, antigen-positive (IE1) cells, the inoculation site in the vaccinees was characterized by an absence of inflammation and antigen-positive cells. All five vaccinees developed robust recall responses to viral antigens, and four of them exhibited long-term viral immune responses consistent with effective control of viral expression and replication. These results demonstrate that a heterologous DNA prime/protein boost strategy greatly expands the breadth of antiviral immune responses and greatly reduces the level of viral replication at the primary site of challenge infection.

Evaluation of recombinant modified vaccinia Ankara virus-based rhesus cytomegalovirus vaccines in rhesus macaques.

A vaccine consisting of rhesus cytomegalovirus (RhCMV) pp65-2, gB and IE1 expressed via modified vaccinia Ankara (MVA) was evaluated in rhesus macaques with or without prior priming with expression plasmids for the same antigens. Following two MVA treatments, comparable levels of anti-gB, pp65-2 and neutralizing antibody responses, and pp65-2- and IE1-specific cellular immune responses were detected in both vaccinated groups. Similar reductions in plasma peak viral loads were observed in these groups compared to untreated controls. This study demonstrates the immunogenicity and protective efficacy of rMVA-based RhCMV subunit vaccines in a primate host and warrants further investigation to improve the efficacy of subunit vaccines against CMV.

Protein coding content of the UL)b' region of wild-type rhesus cytomegalovirus.

A recent comparison of two rhesus cytomegalovirus (RhCMV) genomes revealed that the region at the right end of the U(L) genome component (U(L)b') undergoes genetic alterations similar to those observed in serially passaged human cytomegalovirus (HCMV). To determine the coding content of authentic wild-type RhCMV in this region, the U(L)b' sequence was amplified from virus obtained from naturally infected rhesus macaques without passage in vitro. A total of 24 open reading frames (ORFs) potentially encoding >99 amino acid residues were identified, 10 of which are related to HCMV ORFs and 15 to previously listed RhCMV ORFs. In addition, the analysis revealed a cluster of three novel alpha chemokine-like ORFs, bringing the number of predicted alpha chemokine genes in this region to six. Three of these six genes exhibit a high level of sequence diversity, as has been observed for the HCMV alpha chemokine gene UL146.

Development of breeding populations of rhesus macaques (Macaca mulatta) that are specific pathogen-free for rhesus cytomegalovirus.

Development of breeding colonies of rhesus macaques (Macaca mulatta) that are specific pathogen-free (SPF) for rhesus cytomegalovirus (RhCMV) is relatively straightforward and requires few modifications from current SPF programs. Infants separated from the dam at or within a few days of birth and cohoused with similarly treated animals remain RhCMV seronegative indefinitely, provided they are never directly or indirectly exposed to a RhCMV-infected monkey. By systematically cohousing seronegative animals into larger social cohorts, breeding populations of animals SPF for RhCMV can be established. The additional costs involved in expanding the current definition of SPF status to include RhCMV are incremental compared with the money already being spent on existing SPF efforts. Moreover, the large increase in research opportunities available for RhCMV-free animals arguably would far exceed the development costs. Potential new areas of research and further expansion of existing research efforts involving these newly defined SPF animals would have direct implications for improvements in human health.

Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox.

Approximately 50% of the US population received smallpox vaccinations before routine immunization ceased in 1972 for civilians and in 1990 for military personnel. Several studies have shown long-term immunity after smallpox vaccination, but skepticism remains as to whether this will translate into full protection against the onset of orthopoxvirus-induced disease. The US monkeypox outbreak of 2003 provided the opportunity to examine this issue. Using independent and internally validated diagnostic approaches with >or=95% sensitivity and >or=90% specificity for detecting clinical monkeypox infection, we identified three previously unreported cases of monkeypox in preimmune individuals at 13, 29 and 48 years after smallpox vaccination. These individuals were unaware that they had been infected because they were spared any recognizable disease symptoms. Together, this shows that the US monkeypox outbreak was larger than previously realized and, more importantly, shows that cross-protective antiviral immunity against West African monkeypox can potentially be maintained for decades after smallpox vaccination.

Duration of antiviral immunity after smallpox vaccination.

Although naturally occurring smallpox was eliminated through the efforts of the World Health Organization Global Eradication Program, it remains possible that smallpox could be intentionally released. Here we examine the magnitude and duration of antiviral immunity induced by one or more smallpox vaccinations. We found that more than 90% of volunteers vaccinated 25-75 years ago still maintain substantial humoral or cellular immunity (or both) against vaccinia, the virus used to vaccinate against smallpox. Antiviral antibody responses remained stable between 1-75 years after vaccination, whereas antiviral T-cell responses declined slowly, with a half-life of 8-15 years. If these levels of immunity are considered to be at least partially protective, then the morbidity and mortality associated with an intentional smallpox outbreak would be substantially reduced because of pre-existing immunity in a large number of previously vaccinated individuals.

Complete sequence and genomic analysis of rhesus cytomegalovirus.

The complete DNA sequence of rhesus cytomegalovirus (RhCMV) strain 68-1 was determined with the whole-genome shotgun approach on virion DNA. The RhCMV genome is 221,459 bp in length and possesses a 49% G+C base composition. The genome contains 230 potential open reading frames (ORFs) of 100 or more codons that are arranged colinearly with counterparts of previously sequenced betaherpesviruses such as human cytomegalovirus (HCMV). Of the 230 RhCMV ORFs, 138 (60%) are homologous to known HCMV proteins. The conserved ORFs include the structural, replicative, and transcriptional regulatory proteins, immune evasion elements, G protein-coupled receptors, and immunoglobulin homologues. Interestingly, the RhCMV genome also contains sequences with homology to cyclooxygenase-2, an enzyme associated with inflammatory processes. Closer examination identified a series of candidate exons with the capacity to encode a full-length cyclooxygenase-2 protein. Counterparts of cyclooxygenase-2 have not been found in other sequenced herpesviruses. The availability of the complete RhCMV sequence along with the ability to grow RhCMV in vitro will facilitate the construction of recombinant viral strains for identifying viral determinants of CMV pathogenicity in the experimentally infected rhesus macaque and to the development of CMV as a vaccine vector.

Persistent SIV infection of a blood-brain barrier model.

In order to better model HIV infection of the brain, a dynamic, in vitro model of the blood-brain barrier (the DIV-BBB) was characterized. The model was composed of simian brain microvascular endothelial cells (MVEC) cocultured with human fetal astrocytes under conditions of media flow. Simian immunodeficiency virus (SIV) was introduced into the DIV-BBB model in order to determine whether SIV infection has an effect on the blood-brain barrier (BBB). The cells of the DIV-BBB model were maintained for 127 days, during which a low permeability to sucrose developed. SIV infection of the BBB model was readily accomplished with cell-free virus. Results from ELISA for viral p27 protein, s-MAGI assays, and coculture techniques indicate that SIV productively and persistently infected the BBB model. These studies indicate that SIV can persist in MVEC without overtly compromising BBB function, and suggest that the DIV-BBB will be a highly valuable and suitable model for studies of HIV neuropathogenesis.