A single mutation in chikungunya virus affects vector specificity and epidemic potential

Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005-2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1-226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.     

Assessment of flavaglines as potential chikungunya virus entry inhibitors

Chikungunya virus (CHIKV) is a re‐emerging mosquito‐borne alphavirus that recently caused large epidemics in islands in, and countries around, the Indian Ocean. There is currently no specific drug for therapeutic treatment or for use as a prophylactic agent against infection and no commercially available vaccine. Prohibitin has been identified as a receptor protein used by chikungunya virus to enter mammalian cells. Recently, synthetic sulfonyl amidines and flavaglines (FLs), a class of naturally occurring plant compounds with potent anti‐cancer and cytoprotective and neuroprotective activities, have been shown to interact directly with prohibitin. This study therefore sought to determine whether three prohibitin ligands (sulfonyl amidine 1 m and the flavaglines FL3 and FL23) were able to inhibit CHIKV infection of mammalian Hek293T/17 cells. All three compounds inhibited infection and reduced virus production when cells were treated before infection but not when added after infection. Pretreatment of cells for only 15 minutes prior to infection followed by washing out of the compound resulted in significant inhibition of entry and virus production. These results suggest that further investigation of prohibitin ligands as potential Chikungunya virus entry inhibitors is warranted.     

Transmission potential of chikungunya virus and control measures: the case of Italy

During summer 2007 Italy has experienced an epidemic caused by Chikungunya virus - the first large outbreak documented in a temperate climate country - with approximately 161 laboratory confirmed cases concentrated in two bordering villages in North-Eastern Italy comprising 3,968 inhabitants. The seroprevalence was recently estimated to be 10.2%. In this work we provide estimates of the transmission potential of the virus and we assess the efficacy of the measures undertaken by public health authorities to control the epidemic spread. To such aim, we developed a model describing the temporal dynamics of the competent vector, known as Aedes albopictus, explicitly depending on climatic factors, coupled to an epidemic transmission model describing the spread of the epidemic in both humans and mosquitoes. The cumulative number of notified cases predicted by the model was 185 on average (95% CI 117-278), in good agreement with observed data. The probability of observing a major outbreak after the introduction of an infective human case was estimated to be in the range of 32%-76%. We found that the basic reproduction number was in the range of 1.8-6 but it could have been even larger, depending on the density of mosquitoes, which in turn depends on seasonal meteorological effects, besides other local abiotic factors. These results confirm the increasing risk of tropical vector-borne diseases in temperate climate countries, as a consequence of globalization. However, our results show that an epidemic can be controlled by performing a timely intervention, even if the transmission potential of Chikungunya virus is sensibly high.     

基孔肯雅病毒与基孔肯雅热

基孔肯雅热(chikungunya fever)是由基孔肯雅病毒(chikungunya virus)引起的一种蚊媒传染病,感染率高,可引起持续的关节症状。近几年来,基孔肯雅热暴发次数增加,流行范围不断扩大,全球范围内每年可导致100万人感染。同时,基孔肯雅病毒中某些基因突变使其可有效通过白纹伊蚊传播,不仅对热带和亚热带地区,还对白纹伊蚊广泛存在的温带地区的民众构成了潜在威胁。     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Tonantzitlolones from Stillingia lineata ssp. lineata as potential inhibitors of chikungunya virus

With the purpose of discovering new chemical classes of molecules, in particular those with selective antiviral activity, extracts from plants growing in Reunion Island were systematically evaluated in a chikungunya virus-cell-based assay. The entire ethyl acetate extract obtained from the stem bark of Stillingia lineata ssp. lineata (Euphorbiaceae) exhibited selective antiviral activity against the chikungunya virus with an EC₅₀ < 0.8 μg/mL, whereas only a weak cytotoxic effect was observed on the host cells. A phytochemical investigation of this extract led to the isolation of tonantzitlolone A and tonantzitlolone B (1 and 2), together with the new 4′-hydroxytonantzitlolone, named tonantzitlolone C (3), which has an uncommon C15-flexibilane skeleton, as well as in the new ent-12α-hydroxy-3,7-dioxoisopimara-8,15-diene (4). Subsequent evaluation for inhibition of chikungunya virus replication in cellulo demonstrated that the 4′-acetoxytonantzitlolone (2) was endowed with antiviral activity against CHIKV.     

基孔肯雅病毒疫苗的研究进展

基孔肯雅热是由基孔肯雅病毒(Chikungunya virus,CHIKV)引起的一种急性传染病。基孔肯雅病毒属披膜病毒科甲病毒属。近年来,该病毒死灰复燃,在全球多处引发大规模疫情暴发,是重要的全球性公共卫生问题之一。目前尚无针对CHIKV的疫苗和有效抗病毒药物。疫苗一直是CHIKV研究的热点领域,目前已经取得了很大的进展,就基孔肯雅病毒疫苗的研究进展进行了综述。     

Three-way interactions between mosquito population,viral strain and temperature underlying chikungunya virus transmission potential

Interactions between pathogens and their insect vectors in nature are under the control of both genetic and non-genetic factors, yet most studies on mosquito vector competence for human pathogens are conducted in laboratory systems that do not consider genetic and/or environmental variability. Evaluating the risk of emergence of arthropod-borne viruses (arboviruses) of public health importance such as chikungunya virus (CHIKV) requires a more realistic appraisal of genetic and environmental contributions to vector competence. In particular, sources of variation do not necessarily act independently and may combine in the form of interactions. Here, we measured CHIKV transmission potential by the mosquito Aedes albopictus in all combinations of six worldwide vector populations, two virus strains and two ambient temperatures (20°C and 28°C). Overall, CHIKV transmission potential by Ae. albopictus strongly depended on the three-way combination of mosquito population, virus strain and temperature. Such genotype-by-genotype-by-environment (G × G × E) interactions question the relevance of vector competence studies conducted with a simpler set of conditions. Our results highlight the need to account for the complex interplay between vectors, pathogens and environmental factors to accurately assess the potential of vector-borne diseases to emerge.     

Identification of chikungunya virus interacting proteins in mammalian cells

Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.     

Proteomic analysis of chikungunya virus infected microgial cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms.     

Development of a micro-neutralization test for chikungunya virus

A rapid, micro-scale focus reduction neutralization test for chikungunya virus was developed. In the test, cell monolayers are prepared in a 96-well tissue culture plate and the PAP (peroxidase-antiperoxidase) staining technique is used for detection of foci of chikungunya virus infected cells. This test is suitable for rapid diagnosis and epidemiological studies of the virus.     

Epidemiological investigations of chikungunya epidemic at Barsi, Maharashtra state, India

During the summer of 1973, an epidemic of Chikungunya broke out in Barsi, Sholapur district. Epidemiological investigations carried out during the decline phase revealed that all age groups and both sexes were involved with varying morbidity. The absence of previous immunity was demonstrated by a very high morbidity of 37.53 per cent for the whole town. The clinical picture encountered during the epidemic was quite characteristic of chikungunya. Besides gingivitis and epistaxis there were no frank haemorrhagic episodes. Reports about the presence of demonstrable rash varied. Except for one possible case, there was no mortality as a result of the epidemic. Results of the search for the index case are also described.     

Vertebrate palaeohistology: Past and future

Vertebrate palaeohistology has been considered for a long time as a modest subdivision of Palaeontology. Starting in the 1930s and 1940s, comparative bone tissue histology and palaeohistology progressively demonstrated the multiple correlations between bone tissue distribution and numerous biological variables, such as ontogenetic origin, growth, size, shape, biomechanics, physiology, and ecology. During the last three decades, Palaeohistology has focussed on deciphering the numerous, complex causes explaining the patterns and processes of Vertebrate evolution. Palaeohistology is a powerful tool, in connection with Biology, for the reconstruction of fossil Vertebrates as living organisms.     

Economic botany: Past and future

Economic botany, like botany, had its origin with the herbalists. Its development was encouraged by various European rulers during the Age of Exploration and Discovery in the 16th century. Recent developments in the United States include A. F. Hill’s textbook, Economic Botany, in 1937 the founding of the journal Economic Botany by E. H. Fulling in 1947 and the formation of the Society for Economic Botany in 1959 Some of the breakthroughs in the development of agriculture, beginning with its origin some 10,000 yr ago, are given. It is concluded that we now need another significant breakthrough to feed the world’s population adequately in the year 2000 Some look to genetic engineering to provide such a breakthrough.     

Landscape ecology of sylvatic chikungunya virus and mosquito vectors in southeastern Senegal

The risk of human infection with sylvatic chikungunya (CHIKV) virus was assessed in a focus of sylvatic arbovirus circulation in Senegal by investigating distribution and abundance of anthropophilic Aedes mosquitoes, as well as the abundance and distribution of CHIKV in these mosquitoes. A 1650 km(2) area was classified into five land cover classes: forest, barren, savanna, agriculture and village. A total of 39,799 mosquitoes was sampled from all classes using human landing collections between June 2009 and January 2010. Mosquito diversity was extremely high, and overall vector abundance peaked at the start of the rainy season. CHIKV was detected in 42 mosquito pools. Our data suggest that Aedes furcifer, which occurred abundantly in all land cover classes and landed frequently on humans in villages outside of houses, is probably the major bridge vector responsible for the spillover of sylvatic CHIKV to humans.     

pH-Dependent entry of chikungunya virus fusion into mosquito cells

Background

Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood.

Finding

Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents.

Conclusion

The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.