Human Cytomegalovirus Dysregulates Cellular Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases and Sonic Hedgehog Pathway Proteins in Neural Astrocyte and Placental Models.

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.

Human cytomegalovirus (CMV) dysregulates neurodevelopmental pathways in cerebral organoids.

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD.

Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.

Australian Paediatric Surveillance Unit (APSU) Annual Surveillance Report 2020.

ABSTRACT: For 27 years, national prospective data on selected rare childhood diseases have been collected monthly by the Australian Paediatric Surveillance Unit (APSU) from paediatricians and other clinical specialists who report cases in children aged up to 16 years. We report here the annual results of APSU surveillance in 2020 for ten rare communicable diseases and complications of communicable diseases, namely: acute flaccid paralysis (AFP); congenital cytomegalovirus (CMV) infection; neonatal herpes simplex virus (HSV) infection; perinatal exposure to human immunodeficiency virus (HIV); paediatric HIV infection; severe complications of seasonal influenza; juvenile onset recurrent respiratory papillomatosis (JoRRP); congenital rubella syndrome; congenital varicella syndrome; and neonatal varicella infection. We describe the results for each disease in the context of the total period of study, including demographics, clinical characteristics, treatment and short-term outcomes. Despite challenges presented by the coronavirus disease 2019 (COVID-19) pandemic in 2020, more than 1,400 paediatricians reported regularly to the APSU and an overall monthly reporting rate of > 90% was achieved. The minimum AFP target of 1 case per 100,000 children aged less than 15 years was achieved and there were few cases of vaccine-preventable diseases (JoRRP, rubella, varicella). However, high cases of congenital CMV, neonatal HSV and perinatal exposure to HIV persist. There were no severe complications of seasonal influenza reported for the first time in 13 years. This is consistent with other surveillance data reporting a decline of influenza and other communicable diseases in 2020, and likely reflects the wider effects of public health measures to reduce transmission of SARS-CoV-2 in the Australian community.

Review of studies of severe acute respiratory syndrome related coronavirus-2 pathogenesis in human organoid models.

Severe acute respiratory syndrome related coronavirus-2 (SARS-CoV-2) is the cause of Covid-19 which was classified as a global pandemic in March 2020. The increasing global health and economic burden of SARS-CoV-2 has necessitated urgent investigations into the pathogenesis of disease and development of therapeutic and vaccination regimens. Human trials of vaccine and antiviral candidates have been undertaken, but basic pathogenetic studies are still required to inform these trials. Gaps in understanding of cellular infection by, and immunity to, SARS-CoV-2 mean additional models are required to assist in improved design of these therapeutics. Human organoids are three-dimensional models that contain multiple cell types and mimic human organs in ex vivo culture conditions. The SARS-CoV-2 virus has been implicated in causing not only respiratory injury but also injury to other organs such as the brain, liver and kidneys. Consequently, a variety of different organoid models have been employed to investigate the pathogenic mechanisms of disease due to SARS-CoV-2. Data on these models have not been systematically assembled. In this review, we highlight key findings from studies that have utilised different human organoid types to investigate the expression of SARS-CoV-2 receptors, permissiveness, immune response, dysregulation of cellular functions, and potential antiviral therapeutics.

A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus.

Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.

Serological testing for infectious diseases markers of donor specimens from 24 h after death show no significant change in outcomes from other specimens.

There is increasing demand for organ and tissue donations to cater for a growing waiting list of recipients. Serological screening of donors remains the initial assessment upon which many decisions are made, particularly if donors are found to be seropositive. Multiple different platforms are now available, although the Abbott ARCHITECT platform assays are currently licensed globally for testing of blood collected at less than 15 h post-mortem. Compliance with the specified maximum collection times drastically decreases the number of eligible deceased donors, with ~ 70% more donations available if screened at up to 24 h post mortem. A large scale study on deceased donors was performed where blood was collected between 12 and 25 h post-mortem. A total of 194 cadaveric serological specimens were tested using the Abbott ARCHITECT analyser for Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human T Lymphotropic Virus type I/II, and syphilis infection. The specificity, sensitivity, accuracy, reproducibility and influence of storage conditions were assessed for testing with Abbott ARCHITECT platform for HIV antigen/antibody Combo, HCV antibody, HBV surface antigen (HBsAg), HBV core antibody (HBcAb), HTLVI/II antibody (rHTLV-I/II), and Syphilis TP assays. There was no significant difference between testing of sera from living and cadaveric individuals in terms of assay specificity, sensitivity and accuracy. The findings show testing of human serum and plasma specimens collected up to 24 h post-mortem with these assays is acceptable and reflects host status accurately.

Human cytomegalovirus utilises cellular dual-specificity tyrosine phosphorylation-regulated kinases during placental replication.

INTRODUCTION: Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused indirectly by the placental response to infection. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) have recently been identified as critical kinases for HCMV replication. In this study we provide first evidence that DYRK1A and DYRK1B are utilised during HCMV placental replication. METHODS: DYRK expression was investigated in AD169- and Merlin-infected TEV-1 trophoblast cells, ex vivo placental explants and naturally infected clinical placentae by immunofluorescence, western blot, co-immunoprecipitation and RT-qPCR. RESULTS: HCMV-infected placental cells showed accumulation and re-localisation of DYRK1A and DYRK1B protein to areas of cytoplasmic virion assembly complexes and nuclear viral replication compartments, respectively. This accumulation was a result of upregulated DYRK1A/B protein expression with HCMV inducing up to a 5.3-fold increase in DYRK1A and up to a 4.7-fold increase in DYRK1B protein, relative to mock-infected TEV-1 cells (p < 0.0001). Increased DYRK protein expression was correlated with DYRK1A/B mRNA upregulation, with HCMV-infected cells showing up to a 3.7-fold increase and 2.9-fold increase in DYRK1A and DYRK1B mRNA levels respectively (p < 0.05). Protein-protein interactions were detected between DYRK1A/1B complexes and HCMV immediate early IE2p86, early pp65 and pUL44 and late pp150 proteins. Treatment of HCMV-infected TEV-1 cells and placental explants with DYRK inhibitors significantly inhibited HCMV replication (p < 0.05) indicating these cellular kinases are required during HCMV placental replication. CONCLUSION: HCMV modulates cellular DYRKs during placental replication which may have implications for congenital HCMV pathogenesis and represent promising antiviral targets.

Systematic review of the impact of point-of-care testing for influenza on the outcomes of patients with acute respiratory tract infection.

Acute respiratory tract infections are a major cause of morbidity and mortality and represent a significant burden on the health care system. Laboratory testing is required to definitively distinguish infecting influenza virus from other pathogens, resulting in prolonged emergency department (ED) visits and unnecessary antibiotic use. Recently available rapid point-of-care tests (POCT) may allow for appropriate use of antiviral and antibiotic treatments and decrease patient lengths of stay. We undertook a systematic review to assess the effect of POCT for influenza on three outcomes: (1) antiviral prescription, (2) antibiotic prescription, and (3) patient length of stay in the ED. The databases Medline and Embase were searched using MeSH terms and keywords for influenza, POCT, antivirals, antibiotics, and length of stay. Amongst 245 studies screened, 30 were included. The majority of papers reporting on antiviral prescription found that a positive POCT result significantly increased use of antivirals for influenza compared with negative POCT results and standard supportive care. A positive POCT result also led to decreased antibiotic use. The results of studies assessing the effect of POCT on ED length of stay were not definitive. The studies assessed in this systematic review support the use of POCT for diagnosis of influenza in patients suffering an acute respiratory infection. Diagnosis using POCT may lead to more appropriate prescription of treatments for infectious agents. Further studies are needed to assess the effect of POCT on the length of stay in ED.