Secreted Trimeric Chikungunya Virus Spikes from Insect Cells: Production, Purification, and Glycosylation Status

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies.

The global epidemiology of chikungunya from 1999 to 2020: A systematic literature review to inform the development and introduction of vaccines

Chikungunya fever is an acute febrile illness that is often associated with severe polyarthralgia in humans. The disease is caused by chikungunya virus (CHIKV), a mosquito-borne alphavirus. Since its reemergence in 2004, the virus has spread throughout the tropical world and several subtropical areas affecting millions of people to become a global public health issue. Given the significant disease burden, there is a need for medical countermeasures and several vaccine candidates are in clinical development. To characterize the global epidemiology of chikungunya and inform vaccine development, we undertook a systematic literature review in MEDLINE and additional public domain sources published up to June 13, 2020 and assessed epidemiological trends from 1999 to 2020. Observational studies addressing CHIKV epidemiology were included and studies not reporting primary data were excluded. Only descriptive analyses were conducted. Of 3,883 relevant sources identified, 371 were eligible for inclusion. 46% of the included studies were published after 2016. Ninety-seven outbreak reports from 45 countries and 50 seroprevalence studies from 31 countries were retrieved, including from Africa, Asia, Oceania, the Americas, and Europe. Several countries reported multiple outbreaks, but these were sporadic and unpredictable. Substantial gaps in epidemiological knowledge were identified, specifically granular data on disease incidence and age-specific infection rates. The retrieved studies revealed a diversity of methodologies and study designs, reflecting a lack of standardized procedures used to characterize this disease. Nevertheless, available epidemiological data emphasized the challenges to conduct vaccine efficacy trials due to disease unpredictability. A better understanding of chikungunya disease dynamics with appropriate granularity and better insights into the duration of long-term population immunity is critical to assist in the planning and success of vaccine development efforts pre and post licensure.

Clinical presentations, diagnostics, treatments and treatment costs of children and adults with febrile illness in a tertiary referral hospital in south-eastern Guinea: A retrospective longitudinal cohort study

Background Febrile illness is frequent among patients in the tropics. It is caused by a wide variety of common diseases such as malaria or gastrointestinal infections but also by less common but highly contagious pathogens with epidemic potential. This study describes the clinical features of adult and paediatric patients with febrile illness in in the largest tertiary referral hospital in south-eastern Guinea, a region at high risk for viral haemorrhagic fever outbreaks. The study further compares their diagnostic characteristics, treatments and outcomes with non-febrile patients in order to contribute to the local epidemiology of febrile illness. Methods We used retrospective data collection to record demographic and clinical data of all incoming patients during a study period of three months. For the follow-up study of inpatients, we retrospectively reviewed patient charts for diagnostic characteristics, diagnoses and outcomes. Results Of the 4317 incoming patients during the study period, 9.5% had a febrile illness. The most used diagnostic measures to identify causative agents in febrile patients were point-of-care tests and most treatments relied on antibiotics. Most common discharge diagnoses for febrile inpatients were malaria (9.6% adults, 56.7% children), salmonella gastroenteritis/typhoid (10.6% adults, 7.8% children) and respiratory infection/pneumonia (5.3% adults, 18.7% children). Inpatient mortality for children was significantly higher in febrile than non-febrile children (18.5% vs. 5.1%, p<0.001) and considerably higher in febrile than non-febrile adults (29.8% vs. 25.0%, p = 0.404). Conclusions Malaria, respiratory infection and gastroenteritis are considered the main causes for febrile illness. The wide reliance on rapid diagnostic tests to diagnose febrile patients not only risks to over- or under-diagnose certain diseases but also leaves the possibility of highly infectious diseases in febrile patients unexplored. Furthermore, the heavy reliance on antibiotics risks to cause antimicrobial resistance. High mortality rates in febrile patients, especially children, should be of concern to public health authorities.

Case report of a de novo mutation of PCDH19 in Saudi family limited to females

Up to date more than 60 different mutations in PCDH19 have been identified. Most of PCDH19 gene is located in Xq22 and produces nonclustered delta protocadherin. This disorder primarily manifests in heterozygote females due to random X chromosome inactivation leading to somatic mosaicism and abnormal cellular interference between cells with and without delta-protocadherin., but we a heterozygous nucleotide mutation causing amino acid 561 to change from Pro to Ser (p.Pro561Ser). This mutation was de novo, and this alteration was not found in her parents. PCDH19-related epilepsy is a distinct childhood-onset epilepsy syndrome characterized by brief clusters of febrile and afebrile seizures with onset primarily before the age of three years, cognitive impairment, autistic traits, and behavioral abnormalities. We describe the features of a de novo mutation in 3 sibling, presented with early onset of seizure, two of them were controlled and wean off medication was at age of six year and her sister at age of 10 year .The youngest sister still partially controlled on medication, she had seizure only during febrile illness.

A Clinical Study of the Association of Thrombocytopenia with Acute Febrile Illness

Background: Thrombocytopenia accompanying acute febrile illnesses is a matter of concern because lack of prompt treatment could result in significant mortality. We in this study tried to evaluate the clinical profile of cases with acute fever and thrombocytopenia and determine the cause of fever with thrombocytopenia and the outcome of treatment of such patients in our hospital. Methods: A total of n=50 successive cases of acute febrile illness with thrombocytopenia following inclusion and exclusion criteria were included in this study. Clinical signs such as rashes, signs of dehydration, petechiae, jaundice, lymphadenopathy, hepatomegaly, splenomegaly, anemia, abdominal tenderness, altered sensorium, were noted. Investigations included CBP, ESR, LFT, RFT, serum electrolytes, Chest X-ray, USG abdomen were done. Other investigations included Dengue serology, Malaria, Widal, IgM for leptospirosis, sputum for AFB. Results: Out of n=50 patients with acute fever with thrombocytopenia, all of them had a definitive diagnosis with malaria (40%) as the commonest cause, followed by enteric fever (24%), viral fever (14%), septicemia (6%), dengue (14%), and leptospirosis (2%). 50% of the patients had platelet count in the range of 50, 000 – 1,00, 000 and 30% had platelet counts above 100000-150000. 8% of cases had platelet counts below 25000 and 12% had platelet counts between 25000-50000 at the time of admission. 10% mortality was observed. Conclusion: infections as the commonest cause of thrombocytopenia. Malaria, dengue enteric fever, leptospirosis, and other viral infections formed the major diseases in this group of population. The diagnosis of malaria was the common cause because of seasonal and regional variations. A definitive increase in platelet count was noted after the underlying cause was treated. Severe cases of septicemia with associated co-morbidities resulted in mortality.

Prevalence of Anti-leptospiral IgM and Detection of Pathogenic Leptospira Species DNA in Neonates Presenting With Clinical Sepsis in Southwestern Uganda

BackgroundLeptospirosis is an emerging neglected zoonotic disease that presents with nonspecific signs/symptoms and it can be mistaken for other diseases. Due to limited diagnostic capacity and unawareness, data on human leptospirosis particularly in neonates is scarce in many sub-Saharan countries. It has been underreported hindering preventive and control measures in place. The study aimed at determining prevalence of leptospirosis as a cause of febrile illness in neonates using a commercially available IgM ELISA and a quantitative real-time PCR (qPCR). MethodsThis was a descriptive cross-sectional study that included 103 neonatal sepsis cases whose parents/legal guardians gave informed consent. Data on demographic and clinical characteristics was collected using structured data collection form. EDTA whole blood sample was collected from the neonates by trained study nurses. From the samples, IgM ELISA was done using automated analyzers, DNA extracted and qPCR was performed using primers for LipL32, specific for the pathogenic leptospires. ResultsThe prevalence of anti-leptospiral IgM among the neonates as determined by ELISA was 4.3%, where all of them presented with lethargy and poor feeding. No pathogenic Leptospira species DNA was amplified by qPCR.ConclusionsEvidence of leptospirosis was demonstrated in neonatal sepsis cases in this study. The findings suggest considerations of leptospirosis in the differential diagnosis of neonates with sepsis. More data is needed on the real epidemiology, clinical features and burden of leptospirosis in neonates. There is need to include intermediate pathogenic species of Leptospira in the diagnostic qPCR assays.

Chronic aseptic meningitis caused by enterovirus in a humorally immunosuppressed adult patient presenting with sensorineural hearing loss: a case report

Background Enterovirus has been described as a cause of aseptic meningitis in humorally immunosuppressed patients. Case presentation A 67-year-old female with a history of mantle cell lymphoma on rituximab therapy presented with subacute hepatitis, myalgias, and sensorineural hearing loss several months after an initial febrile illness. She was diagnosed with enterovirus infection by CSF PCR as a unifying etiology of her presentation, representing an unusual presentation of disease. Discussion and conclusions This patient’s unique presentation and clinical course presents important implications in the care of similarly immunosuppressed patients with cryptic complaints.

Urticarial eruption in COVID-19-positive children: A report of two cases

Recent literature has reported a variety of dermatological manifestations in children and adults associated with COVID-19. Herein, we report urticarial eruptions in two COVID-19-positive children. In the first case, urticaria with angioedema preceded a febrile episode and only partially responded to conventional doses of antihistamines. In the second case, urticaria followed the appearance of fever and upper respiratory symptoms. Both cases recovered completely within two weeks of diagnosis. These cases demonstrate that urticaria and angioedema, precedent or following a febrile illness, with or without respiratory symptoms, may be a presenting symptom of COVID-19 infection in children. A high index of suspicion in such cases helps the early administration of treatment and isolation of the patients to limit the spread of the virus.