Development of a Novel Plasmid-based Eukaryotic Model to Investigate Crimean-Congo Hemorrhagic Fever Virus

Objective: Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne viral disease, caused by the Crimean-Congo Hemorrhagic Fever virus (CCHFV). The global expansion of CCHF and high mortality rates underline the critical need for research and development of effective treatments and vaccines. However, the high risk of transmission and requirement for highcontainment facilities hinder investigations involving live virus. In this study, we aimed to address these challenges by employing a plasmid-based virus-like particle (VLP) system and a minigenome model to investigate the biology and immunology of CCHFV. Methods: The plasmids encoding CCHFV structural genes of CCHFV were transfected into Huh-7 cells. Viral protein expression was confirmed using fluorescence imaging, immunological and molecular methods. A minigenome system was developed, eliminating the need for T7 polymerase, T7-expressing cellular lines, or viral ribonuclear protein complexes, allowing autonomous virus replication without a helper virus or transfections using plasmids in trans. Results: Fluorescence microscopy showed successful production of NP-EGFP and GC-EGFP proteins with various subcellular localizations. Western blot analysis demonstrated the presence of pre-Gc, Gc, pre-Gn, Gn, and Np proteins in cell lysates and supernatants. ELISA analysis suggested that transfection of Np alone, in combination with Gc, or all three proteins might cause distinct VLP formations. Huh-7 cells successfully expressed reporter genes after transfection of minigenome RNA transcripts. Conclusion: The study advances CCHFV research by using novel tools for virus biology and immunology. The findings may provide new avenues for research that promise better public health preparation against this neglected viral disease

Cross-Reactive Anti-Nucleocapsid Protein Immunity against Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus in Multiple Species

The World Health Organization estimates that there may be three billion people at risk of infection by Crimean-Congo hemorrhagic fever virus (CCHFV), a highly lethal emerging Orthonairovirus carried by ticks. On the other hand, the closely related Hazara virus (HAZV), a member of the same serogroup, has not been reported as a pathogen for humans. Given the structural and phylogenetic similarities between these two viruses, we evaluated the immunological similarities of the nucleocapsid protein (NP) of these two viruses in multiple species. Strong antigenic similarities were demonstrated in anti-NP humoral immune responses against HAZV and CCHFV in multiple species using convalescent-phase human CCHF sera, rabbit and mouse polyclonal antiserum raised against CCHFV, and mouse polyclonal antiserum against CCHFV-NP in enzyme immunoassays. We also report a convincing cross-reactivity between NPs in Western blots using HAZV-infected cell lysate as antigen and inactivated CCHFV- and CCHFV-NP-immunized mouse sera. These results suggest that NPs of HAZV and CCHFV share significant similarities in humoral responses across species and underline the potential utility of HAZV as a surrogate model for CCHFV

Long-term follow-up of alkaptonuria patients: single center experience

Objectives Alkaptonuria is a rare autosomal recessive genetic disorder resulting from the deficiency of homogentisate 1,2 dioxygenase (HGD), the third enzyme in the tyrosine degradation pathway. Homogentisic acid produced in excess oxidizes into ochronotic pigment polymer. Accumulation of this pigment in various tissues leads to systemic disease. Methods Clinical, laboratory, molecular findings and treatment characteristics of 35 patients followed up in Ege University Pediatric Nutrition, and Metabolism Department with the diagnosis of alkaptonuria were evaluated retrospectively. Results Twenty-four males (68.57%) and 11 females (31.42%) with a confirmed diagnosis of alkaptonuria from 32 different families were included in the study. We identified 11 different genetic variants; six of these were novel. c.1033C>T, c.676G>A, c.664G>A, c.731_734del, c.1009G>T, c.859_862delins ATAC were not previously reported in the literature. 24 (68.57%) patients only adhered to a low-protein diet in our study group. Seven (20%) patients initiated a low protein diet and NTBC therapy. Mean urinary HGA decreased by 88.7% with nitisinone. No statistical changes were detected in urinary HGA excretion with the low protein diet group. Conclusions In our study, alkaptonuria patients were diagnosed at different ages, from infancy to adulthood, and progressed with other systemic involvement in the follow-up. Since the initial period is asymptomatic, giving potentially effective treatment from an early age is under discussion. Raising disease awareness is very important in reducing disease mortality and morbidity rates

An Attenuated HSV-1-Derived Malaria Vaccine Expressing Liver-Stage Exported Proteins Induces Sterilizing Protection against Infectious Sporozoite Challenge

Here, we present the construction of an attenuated herpes simplex virus type-1 (HSV-1)-vectored vaccine, expressing three liver-stage (LS) malaria parasite exported proteins (EXP1, UIS3 and TMP21) as fusion proteins with the VP26 viral capsid protein. Intramuscular and subcutaneous immunizations of mice with a pooled vaccine, composed of the three attenuated virus strains expressing each LS antigen, induced sterile protection against the intravenous challenge of 17X-NL salivary gland sporozoites. Our data suggest that this malaria vaccine may be effective in preventing malaria parasite infection using practical routes of immunization in humans

An Attenuated HSV-1-Derived Malaria Vaccine Expressing Liver-Stage Exported Proteins Induces Sterilizing Protection against Infectious Sporozoite Challenge

Here, we present the construction of an attenuated herpes simplex virus type-1 (HSV-1)-vectored vaccine, expressing three liver-stage (LS) malaria parasite exported proteins (EXP1, UIS3 and TMP21) as fusion proteins with the VP26 viral capsid protein. Intramuscular and subcutaneous immunizations of mice with a pooled vaccine, composed of the three attenuated virus strains expressing each LS antigen, induced sterile protection against the intravenous challenge of Plasmodium yoelii 17X-NL salivary gland sporozoites. Our data suggest that this malaria vaccine may be effective in preventing malaria parasite infection using practical routes of immunization in humans

Tetrahydrobiopterin deficiencies: Lesson from clinical experience

Objectives The present study describes clinical, biochemical, molecular genetic data, current treatment strategies and follow-up in nine patients with tetrahydrobiopterin (BH4) deficiency due to various inherited genetic defects. Methods We analyzed clinical, biochemical, and molecular data of nine patients with suspected BH4 deficiency. All patients were diagnosed at Ege University Faculty of Medicine in Izmir, Turkey and comprised data collected from 2006 to 2019. The diagnostic laboratory examinations included blood phenylalanine and urinary or plasma pterins, dihydropteridine reductase (DHPR) enzyme activity measurement in dried blood spots, folic acid and monoamine neurotransmitter metabolites in cerebrospinal fluid, as well as DNA sequencing. Results Among the nine patients, we identified one with autosomal recessive GTP cyclohydrolase I (ar GTPCH) deficiency, two with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, three with sepiapterin reductase (SR) deficiency, and three with DHPR deficiency. Similar to previous observations, the most common clinical symptoms are developmental delay, intellectual disability, and movement disorders. All patients received treatment with l-dopa and 5-hydroxytryptophan, while only the ar GTPCH, the PTPS, and one DHPR deficient patients were supplemented in addition with BH4. The recommended dose range varies among patients and depends on the type of disease. The consequences of BH4 deficiencies are quite variable; however, early diagnosis and treatment will improve outcomes. Conclusions As BH4 deficiencies are rare group of treatable neurometabolic disorders, it is essential to diagnose the underlying (genetic) defect in newborns with hyperphenylalaninemia. Irreversible brain damage and progressive neurological deterioration may occur in untreated or late diagnosed patients. Prognosis could be satisfying in the cases with early diagnose and treatment

Subacute THYROiditis Related to SARS-CoV-2 VAccine and Covid-19 (THYROVAC Study): A Multicenter Nationwide Study.

Context The aims of the study are to compare characteristics of subacute thyroiditis (SAT) related to different etiologies, and to identify predictors of recurrence of SAT and incident hypothyroidism. Methods This nationwide, multicenter, retrospective cohort study included 53 endocrinology centers in Turkey. The study participants were divided into either COVID-19-related SAT (Cov-SAT), SARS-CoV-2 vaccine-related SAT (Vac-SAT), or control SAT (Cont-SAT) groups. Results Of the 811 patients, 258 (31.8%) were included in the Vac-SAT group, 98 (12.1%) in the Cov-SAT group, and 455 (56.1%) in the Cont-SAT group. No difference was found between the groups with regard to laboratory and imaging findings. SAT etiology was not an independent predictor of recurrence or hypothyroidism. In the entire cohort, steroid therapy requirement and younger age were statistically significant predictors for SAT recurrence. C-reactive protein measured during SAT onset, female sex, absence of antithyroid peroxidase (TPO) positivity, and absence of steroid therapy were statistically significant predictors of incident (early) hypothyroidism, irrespective of SAT etiology. On the other hand, probable predictors of established hypothyroidism differed from that of incident hypothyroidism. Conclusion Since there is no difference in terms of follow-up parameters and outcomes, COVID-19- and SARS-CoV-2 vaccine-related SAT can be treated and followed up like classic SATs. Recurrence was determined by younger age and steroid therapy requirement. Steroid therapy independently predicts incident hypothyroidism that may sometimes be transient in overall SAT and is also associated with a lower risk of established hypothyroidism