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.

BackgroundChikungunya (CHIK) virus is a mosquito-transmitted alphavirus that causes in humans an acute infection characterised by fever, polyarthralgia, head-ache, and myalgia. Since 2005, the emergence of CHIK virus was associated with an unprecedented magnitude outbreak of CHIK disease in the Indian Ocean. Clinically, this outbreak was characterized by invalidating poly-arthralgia, with myalgia being reported in 97.7% of cases. Since the cellular targets of CHIK virus in humans are unknown, we studied the pathogenic events and targets of CHIK infection in skeletal muscle.Methodology/Principal FindingsImmunohistology on muscle biopsies from two CHIK virus-infected patients with myositic syndrome showed that viral antigens were found exclusively inside skeletal muscle progenitor cells (designed as satelllite cells), and not in muscle fibers. To evaluate the ability of CHIK virus to replicate in human satellite cells, we assessed virus infection on primary human muscle cells; viral growth was observed in CHIK virus-infected satellite cells with a cytopathic effect, whereas myotubes were essentially refractory to infection.Conclusions/SignificanceThis report provides new insights into CHIK virus pathogenesis, since it is the first to identify a cellular target of CHIK virus in humans and to report a selective infection of muscle satellite cells by a viral agent in humans.

A mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile (WN) virus, a member of the genus flavivirus and family Flaviviridae. Whereas WN virus causes encephalitis and death in most laboratory inbred mouse strains after peripheral inoculation, most strains derived from recently trapped wild mice are completely resistant. The phenotype of resistance͞susceptibility is determined by a major locus, Wnv, mapping to chromosome 5 within the 0.4-cM-wide interval defined by markers D5Mit408 and D5Mit242. We constructed a high resolution composite͞consensus map of the interval by merging the data from the mouse T31 Radiation Hybrid map and those from the homologous region of human chromosome 12q, and found the cluster of genes encoding 2 -5 -oligoadenylate synthetases (2 -5 -OAS) to be the most prominent candidate. This cluster encodes a multimember family of IFN-inducible proteins that is known to play an important role in the established endogenous antiviral pathway. Comparing the cDNA sequences of 2 -5 -OAS L1, L2, and L3 isoforms, between susceptible and resistant strains, we identified a STOP codon in exon 4 of the gene encoding the L1 isoform in susceptible strains that can lead to a truncated form with amputation of one domain, whereas all resistant mice tested so far have a normal copy of this gene. The observation that WN virus sensitivity of susceptible mice was completely correlated with the occurrence of a point mutation in 2 -5 -OAS L1 suggests that this isoform may play a critical role in WN pathogenesis.F lavivirus is a genus of the Flaviviridae family that comprises over 70 positive-sense, single-stranded, and enveloped RNA viruses, most of which are arthropod-borne (1). Mosquito-borne flaviviruses such as dengue (DEN), Japanese encephalitis (JE), yellow fever (YF), and West Nile (WN) viruses can cause epidemic outbreaks in humans, and patients infected may exhibit a wide range of acute diseases, from nonspecific febrile illness to severe hemorrhagic manifestations (DEN and YF) or encephalitic syndromes (JE and WN), sometimes associated with a high (up to 50%) fatality rate. The reasons why flaviviruses cause clinical manifestations only in a small percentage of infected individuals have not yet been clearly elucidated but are supposed to involve host-dependent genetic factors.Over the past 5 years, WN fever has been an emerging concern for public health in Europe, in the Middle East, and more recently in the United States. In Israel and the United States, where the Isr98͞NY99 variant of WN virus was frequently isolated during recent outbreaks, approximately 20% of infected persons developed febrile illness, with a high rate of mortality among patients with neurological symptoms (2, 3). This result suggests that susceptibility and sensitivity to WN virus infections might depend, at least in part, on host genetic factors (4). In the present study we investigated the influence of the host genetic constitution on the severity of WN virus infection, by using...

Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.Members of the Lyssavirus genus are nonsegmented negative-strand RNA viruses belonging to the Mononegavirales order, Rhabdoviridae family. On the basis of phylogenetic studies, seven genotypes have been distinguished among which genotype 1 (rabies virus, PV strain) and genotype 3 (Mokola virus) are the most divergent (5, 44). These enveloped viruses are responsible for rabies encephalomyelitis. Usually transmitted mechanically by bite, injury, or aerosol, lyssaviruses are highly neurotropic, migrating from inoculation point to the central nervous system (CNS) through peripheral nerves. Their viral cycle takes place in the cytoplasm, where the viral genetic information exclusively found in the form of a ribonucleoprotein (RNP) complex serves as a template for two distinct RNA synthesis functions: transcription of a leader RNA and 5Ј capped and polyadenylated mRNAs encoding the different viral proteins (nucleoprotein [N], phosphoprotein [P], matrix protein [M], glycoprotein [G], and RNA polymerase [L]) and viral replication occurring in anti-genomic and new genomic RNA molecule synthesis. Transcription and replication are insured by the RNP complex composed of the L protein associated with the P protein and the genomic RNA tightly enwrapped by the N protein. The P protein via N:P complexes prevents nonspecific N protein aggregation while the L protein, considered the catalytic core, attaches to the N:RNA template through interactions with P. Thus, the P protein is considered to play a dual and pivotal role in this regulation. The P protein (297 amino acids [aa], PV strain [genotype 1]; 303 aa, Mokola virus [genotype 3]) is thought to be composed of two conserved domains: one is NH 2 terminal (the first 60 aa residue...

Chikungunya virus (CHIKV) is a mosquito-transmittedAlphavirus that causes in humans an acute infection characterized by polyarthralgia, fever, myalgia, and headache. Since 2005 this virus has been responsible for an epidemic outbreak of unprecedented magnitude. By analogy with other alphaviruses, it is thought that cellular proteases are able to process the viral precursor protein E3E2 to produce the receptorbinding E2 protein that associates as a heterodimer with E1. Destabilization of the heterodimer by exposure to low pH allows viral fusion and infection. We show that among a large panel of proprotein convertases, membranous furin but also PC5B can process E3E2 from African CHIKV strains at the HRQRR 64 2ST site, whereas a CHIKV strain of Asian origin is cleaved at RRQRR 64 2SI by membranous and soluble furin, PC5A, PC5B, and PACE4 but not by PC7 or SKI-1. Using fluorogenic model peptides and recombinant convertases, we observed that the Asian strain E3E2 model peptide is cleaved most efficiently by furin and PC5A. This cleavage was also observed in CHIKV-infected cells and could be blocked by furin inhibitor decanoyl-RVKR-chloromethyl ketone. This inhibitor was compared with chloroquine for its ability to inhibit CHIKV spreading in myoblast cell cultures, a cell-type previously described as a natural target of this virus. Our results demonstrate the role of furin-like proteases in the processing of CHIKV particles and point out new approaches to inhibit this infection. Chikungunya virus (CHIKV)4 is a mosquito-transmitted Alphavirus belonging to the family Togaviridae, which was first reported in 1952 in Tanganyika. It is responsible for an acute infection of abrupt onset characterized by high fever, polyarthralgia, myalgia, headache, chills, and rash (1). The symptoms are generally of short duration (1 week), and recovery is often complete, although some patients have recurrent episodes for several weeks after infection (1, 2). CHIKV is endemic in Africa, India, and Southeast Asia and is transmitted by Aedes mosquitoes through an urban or sylvatic transmission cycle. In 2006 an outbreak of CHIKV fever occurred in numerous islands of the Indian Ocean (the Comoros, Mauritius, Seychelles, Madagascar, La Réunion, etc.) before jumping to India where an estimated 1.4 million cases have been reported (3-5). More recently, imported infections have been described in Europe, and around 200 endemic cases have been reported in Italy (6). Clinically, this CHIKV epidemic was accompanied by more severe symptoms than previous outbreaks, with reports of severe polyarthralgia and myalgia, complications, and deaths. In muscle biopsies from two infected adults we could identify cellular targets for CHIKV as muscle satellite cells (the muscle progenitor cells) and confirmed in vitro the susceptibility of these cells to CHIKV infection (7). Up until now no other cell targets have been identified in humans except for a recent report showing that fibroblast cells were infected in a fatal neonatal case (8). Other cell types such as ...

Rabies virus (RV), a highly neurotropic enveloped virus, is known to spread within the CNS by means of axonal transport. Although the envelope spike glycoprotein (G) of cell-free virions is required for attachment to neuronal receptors and for virus entry, its necessity for transsynaptic spread remains controversial. In this work, a G gene-deficient recombinant RV (SAD ∆G) complemented phenotypically with RV G protein (SAD ∆GMG) has been used to demonstrate the absolute requirement for G in virus transfer from one neuron to another, both in vitro, in neuronal cell cultures (cell line and primary cultures), and in vivo, in murine animal models. By using a model of stereotaxic inoculation into the rat striatum, infection is shown to be restricted to initially infected cells and not transferred to secondary neurons. In mouse as in rat models of infection, the limited infection did not cause any detectable symptoms, suggesting that Gdeficient RV recombinants might be valuable as non-pathogenic, single-round vectors for expression of foreign genes.

Abstract. Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2÷/calmodulin-dependent protein kinase II (CaM kinase ]I). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.

This review provides for the first time an assessment of the current understanding about the occurrence and the clinical significance of gastrointestinal (GI) symptoms in influenza patients, and their correlation with the presence of human influenza viruses in stools of patients with confirmed influenza virus infection. Studies exploring how human influenza viruses spread to the patient’s GI tract after a primary respiratory infection have been summarized. We conducted a systematic search of published peer-reviewed literature up to June 2015 with regard to the above-mentioned aspects, focusing on human influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), and B). Forty-four studies were included in this systematic review and meta-analysis. The pooled prevalence of any digestive symptoms ranged from 30.9 % (95 % CI, 9.8 to 57.5; I2 = 97.5 %) for A(H1N1)pdm09 to 2.8 % (95 % CI, 0.6 to 6.5; I2 = 75.4 %) for A(H1N1). The pooled prevalence of influenza viruses in stool was 20.6 % (95 % CI, 8.9 to 35.5; I2 = 96.8 %), but their correlation with GI symptoms has rarely been explored. The presence of viral RNA in stools because of haematogenous dissemination to organs via infected lymphocytes is likely, but the potential to cause direct intestinal infection and faecal–oral transmission warrants further investigation. This review highlights the gaps in our knowledge, and the high degree of uncertainty about the prevalence and significance of GI symptoms in patients with influenza and their correlation with viral RNA positivity in stool because of the high level of heterogeneity among studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0448-4) contains supplementary material, which is available to authorized users.