Expression of the capsid protein of Chikungunya virus in a baculovirus for serodiagnosis of Chikungunya disease

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIK fever, which typically manifests as a febrile illness. To develop a CHIKV-specific diagnostic test, CHIKV capsid protein was expressed using a baculovirus expression system. The seroreactvity of the recombinant CHIKV capsid protein was evaluated by ELISA and immuochromatographic assay (ICA), using 40 anti-CHIKV-positive and 20 anti-CHIKV-negative sera, an additional 20 normal sera samples from healthy Koreans, and 20 anti-Dengue virus sera samples. The sensitivity of the recombinant CHIKV capsid protein was 85% and 87.5% as measured by ELISA and ICA, respectively. The specificity of the recombinant CHIKV capsid protein was 100% both by ELISA and by ICA. No cross-reactivity of the capsid protein was seen with anti-Dengue virus sera samples. There was a significant correlation between the ELISA- and ICA-measured seroreactivities of the recombinant CHIKV capsid protein for anti-CHIKV IgM-positive sera samples. These results suggest that the recombinant CHIKV capsid protein could be used in a diagnostic test for identifying CHIKV disease.     

Outcomes of persons with coronavirus disease 2019 in hospitals with and without standard treatment with (hydroxy)chloroquine

ObjectiveTo compare survival of individuals with coronavirus disease 2019 (COVID-19) treated in hospitals that either did or did not routinely treat patients with hydroxychloroquine or chloroquine.MethodsWe analysed data of COVID-19 patients treated in nine hospitals in the Netherlands. Inclusion dates ranged from 27 February to 15 May 2020, when the Dutch national guidelines no longer supported the use of (hydroxy)chloroquine. Seven hospitals routinely treated patients with (hydroxy)chloroquine, two hospitals did not. Primary outcome was 21-day all-cause mortality. We performed a survival analysis using log-rank test and Cox regression with adjustment for age, sex and covariates based on premorbid health, disease severity and the use of steroids for adult respiratory distress syndrome, including dexamethasone.ResultsAmong 1949 individuals, 21-day mortality was 21.5% in 1596 patients treated in hospitals that routinely prescribed (hydroxy)chloroquine, and 15.0% in 353 patients treated in hospitals that did not. In the adjusted Cox regression models this difference disappeared, with an adjusted hazard ratio of 1.09 (95% CI 0.81–1.47). When stratified by treatment actually received in individual patients, the use of (hydroxy)chloroquine was associated with an increased 21-day mortality (HR 1.58; 95% CI 1.24–2.02) in the full model.ConclusionsAfter adjustment for confounders, mortality was not significantly different in hospitals that routinely treated patients with (hydroxy)chloroquine compared with hospitals that did not. We compared outcomes of hospital strategies rather than outcomes of individual patients to reduce the chance of indication bias. This study adds evidence against the use of (hydroxy)chloroquine in hospitalised patients with COVID-19.     

The Chikungunya virus

Chikungunya virus (CHIKV), a member of the Alphavirus genus, represents a real public health problem in tropical regions of the Southeast Asia and Africa. It is transmitted to the man by Aedes mosquitoes and the illness, known as Chikungunya, is characterized by fever, eruptions and invalidating arthralgia. An increased surveillance in tropical and subtropical areas is necessary, as far as we have noticed recently the emergence of this new disease in regions where it had never existed before. The epidemic context is of a high importance for diagnosis. It is very important to know the clinical characteristics of the infection, to detect forms rarely or never described previously. Permanence of a highly technical core in specialized laboratories will allow, fast, specific and differential diagnosis. The knowledge of the epidemiological chain of transmission from reservoir, still unknown, to the host aims to protect populations by limiting the risks of exposure when it is possible. The only prevention measures available are individual protection against mosquitoes and antivectorial fight, in the absence of specific antiviral treatment and vaccine.     

Mouse models for Chikungunya virus: deciphering immune mechanisms responsible for disease and pathology

Chikungunya virus (CHIKV), an alphavirus, has been responsible for large epidemic outbreaks with serious economic and social impact during the last 6?years. Transmitted by Aedes mosquitoes, it causes Chikungunya fever, an acute illness in patients with a stooped posture often associated with chronic and incapacitating arthralgia. The unprecedented re-emergence has stimulated renewed interest in CHIKV. This review discusses the advantages and disadvantages of different animal models for CHIKV infections and their importance to study the role of the immune system in different pathologies caused by CHIKV. We also reveal how such studies still present a difficult challenge, but are indispensible for mechanistic studies to further understand the pathophysiology of CHIKV infections.     

Exocytosis of intact lysosomes from skeletal muscle after chloroquine treatment

Following short-term chloroquine treatment (25 mg/kg daily for 7 days), intact myeloid bodies were extruded from the anterior latissimus dorsi (ALD) muscle of the chicken. These myeloid bodies demonstrated acid phosphatase activity both within the cell and after extrusion into the extracellular space. Tannic acid staining confirmed that the plasma membrane remained intact and the “extracellular lysosomes” observed had not resulted from fixation damage. Lysosome extrusion following overloading in skeletal muscle and the fate of the lysosomal contents after extrusion are discussed.     

Chikungunya, the Traveling Virus

Chikungunya virus is an arbovirus transmitted principally by the Aedes mosquitoes. An infection with the virus causes fever, severe arthralgias, and rash. The virus caused a large outbreak on the island of Reunion in the Indian Ocean in 2006, along with an outbreak in India. One of its vectors, Aedes albopictus, has established itself in southern Europe and the United States. With the vector in place, it was feared that outbreaks of chikungunya could occur outside the tropics in temperate climates. Travelers returning to Italy provided the source of virus in the summer of 2007, resulting in the first outbreak of this disease in Europe. This review will discuss the history of chikungunya infections, the roles of its vectors and globalization in its transmission, and preventative measures to control the effects of the virus.     

Stage independent chloroquine resistance and chloroquine toxicity revealed via spinning disk confocal microscopy

We previously customized a Nipkow spinning disk confocal microscope (SDCM) to acquire 4D data for live, intraerythrocytic malarial parasites [Gligorijevic B, McAllister R, Urbach JS, Roepe, PD. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria. Biochemistry 2006;45:12400-10]. We reported that chloroquine (CQ) treatment did not appear to affect progress through the cell cycle, and suggested that toxicity may be manifested post-schizogony. We now use SDCM, synchronized cell culture and continuous vs. bolus drug dosing to investigate stage specific CQ effects in detail. We develop a novel, extremely rapid method for counting schizont nuclei in 3D. We then quantify schizont nuclei and hemozoin (Hz) production for live parasite cultures pulsed with CQ at different stages in the cell cycle and find that bolus treatment of rings affects the multiplicity of nuclear division. We quantify parasitemia and merozoite development in subsequent cycles following bolus CQ exposure and find that a portion of CQ toxicity is manifested post-schizogony as "delayed death". Using these methods and others we compare CQ sensitive (CQS) vs. resistant (CQR) strains as well as transfectants that are CQR via introduction of mutant PfCRT. Surprisingly, we find that PfCRT confers resistance to CQ administered at the very early ring stage of development, wherein a digestive vacuole is not yet formed, as well as at the schizont stage, wherein Hz production is thought to plateau. Taken together, these data force a rethinking of CQ pharmacology and the mechanism of CQR.     

Expression and evaluation of Chikungunya virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection

Purpose

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens.

Materials and Methods

CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA.

Results

The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%.

Conclusion

The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.     

Molecular and serological diagnosis of Chikungunya virus infection

INTRODUCTION: In 2005-2006, during the Chikungunya virus outbreak in La Réunion (Indian Ocean), we urgently established the molecular and serological methods for the diagnosis of Chikungunya virus (CHIKV) from various types of samples. METHODS: CHIKV RNA was detected using a highly sensitive real-time RT PCR assay. A co-extracted and co-amplified internal control RNA was used to identify RT PCR inhibitors. Depending on their nature samples were pretreated before nucleic acid extraction. Viral loads were measured using a synthetic RNA calibrator. CHIKV immunoglobulin (Ig) G and M antibodies were detected by ELISA either from sera or from blood absorbed on filter paper. RESULTS: CHIKV RNA was found in various types of samples such as plasma, cerebrospinal fluid, and placenta, but was not found in some samples including maternal milk and synovial samples. Detection of IgG from filter paper absorbed blood is specific and sensitive. Routine data showed that maternally transferred IgG and naturally acquired IgM persist at least 12 and 18 months, respectively. DISCUSSION: The techniques enabled the diagnosis of chikungunya in known and newly described forms of the disease. They are used for routine diagnosis and large scale surveys.     

氯喹对地塞米松抑制急性淋巴细胞白血病细胞增殖的促进作用*

 目的：研究氯喹对地塞米松抑制急性淋巴细胞白血病细胞增殖的促进作用及其机制。方法：采用CCK-8实验观察氯喹对地塞米松抑制人急性淋巴细胞白血病细胞株CEM-C1增殖的促进作用,并采用Western blotting、实时定量PCR和LysoTracker Red溶酶体染色法验证此种促进作用的机制。结果：氯喹与地塞米松合用对CEM-C1细胞增殖的抑制明显高于正常对照组（P<0.01）。氯喹与地塞米松联用上调糖皮质激素受体（GR）的蛋白质水平并抑制溶酶体功能,而溶酶体抑制剂bafilomycin A1亦可上调GR的蛋白质水平。结论：氯喹与地塞米松联用通过溶酶体介导的机制发挥抑制急性淋巴细胞白血病细胞增殖的作用。     

The characterization of Chikungunya virus RNA

Summary Highly purified preparations of Chikungunya virus (CHV) were obtained from culture fluid of infected VERO cells (Green monkey kidney stable cells) by differential centrifugation involving sedimentation through 25% sucrose solution and density gradient centrifugation by the use of potassium tartrate (17 to 27 %). When the suspension of³²P labeled purified CHV was fractionated with acid,³²P was recovered mostly from the ribonucleic acid (RNA) and the lipid fraction. The base composition (per 100 nucleotides) of CHV-RNA was U=23.5, G=21.0, C=22.5, and A=33.0.Intact RNA molecules in an extended form were extracted from purified CHV virions with a mixture of sodium dodecyl sulfate (SDS) and trypsin. Rotary shadowed preparations were examined in the electron microscope. The length distribution of the RNA molecules showed a prominent peak between 2.4 and 2.8 , giving a modal length of 2.58 . The molecular weight of CHV-RNA was estimated as 2.6×10⁶ daltons.