Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC₅₀) or effective concentration (EC₅₀), as well as its range of cytotoxicity (CC₅₀). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.     

Discovery of a Novel Compound with Anti-Venezuelan Equine Encephalitis Virus Activity That Targets the Nonstructural Protein 2

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC₅₀ = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.     

A Herpes Simplex Virus Scaffold Peptide That Binds the Portal Vertex Inhibits Early Steps in Viral Replication

Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.     

肠道病毒、乙脑病毒和腮腺炎病毒多重RT-PCR检测方法的建立

为建立针对肠道病毒(enterovirus,EV)、乙脑病毒(Japanese encephalitis virus,JEV)和腮腺炎病毒(mumps virus,MUV)的多重RT-PCR检测方法,分别选择肠道病毒的5′UTR基因、乙脑病毒的E基因和腮腺炎病毒M基因设计3对引物,建立同时检测肠道病毒、乙脑病毒和腮腺炎病毒的多重RT-PCR方法.以中国地区流行的与脑炎相关的麻疹病毒和风疹病毒cDNA为模板验证该检测方法的特异性,同时以梯度稀释的不同滴度的脊髓灰质炎病毒、乙脑病毒、腮腺炎病毒评估该检测方法的检出限.所建立的3种病毒的多重RT-PCR方法可同时或分别特异扩增肠道病毒、乙脑病毒和腮腺炎病毒的152 bp、429 bp和274 bp基因片段,基因序列分别与脊髓灰质炎病毒Sabin 1型5′UTR基因片段、乙型脑炎病毒SA-14-14-2株E基因片段及腮腺炎病毒S79基因片段序列一致;检出限分别达到78.1 CCID50/mL、312.5 PFU/mL、156.2 CCID50/mL;而对麻疹病毒和风疹病毒的扩增均为阴性.所建立的多重RT-PCR特异性和检出限良好,可用于上述3种脑炎病毒的快速检测.     

Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.     

Trypsinized Human Group O Erythrocytes as an Alternative Hemagglutinating Agent for Japanese Encephalitis Virus

Trypsinized human group O erythrocytes were found to be a suitable alternative to gander cells in hemagglutination (HA) and hemagglutination inhibition (HAI) tests for Japanese encephalitis (JE) virus. In the HAI test, no cross-reactions against JE virus were observed with immune sera containing antibody to taxonomically related or unrelated viruses, with mouse brain antigen, or with nonantibody serum inhibitors; specific antibody rise could be detected in an immunized rabbit. Gander and trypsinized human group O cells gave comparable titers in the HAI test, but the latter were preferable since (i) they required less challenging HA antigen, being more sensitive to agglutination by JE virus, and (ii) all human and some animal sera investigated were devoid of natural agglutinins for these cells, thereby eliminating or reducing the need for prior adsorption with packed cells.     

C19orf66 Inhibits Japanese Encephalitis Virus Replication by Targeting -1 PRF and the NS3 Protein

Virologica Sinica - The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1′ produced through a programmed -1...     

Involvement of Ceramide in the Propagation of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in the medical and veterinary fields. Although cholesterol has been shown to participate in both the entry and replication steps of JEV, the mechanisms of infection, including the cellular receptors of JEV, remain largely unknown. To clarify the infection mechanisms of JEV, we generated pseudotype (JEVpv) and recombinant (JEVrv) vesicular stomatitis viruses bearing the JEV envelope protein. Both JEVpv and JEVrv exhibited high infectivity for the target cells, and JEVrv was able to propagate and form foci as did authentic JEV. Anti-JEV envelope antibodies neutralized infection of the viruses. Treatment of cells with inhibitors for vacuolar ATPase and clathrin-mediated endocytosis reduced the infectivity of JEVpv, suggesting that JEVpv enters cells via pH- and clathrin-dependent endocytic pathways. Although treatment of the particles of JEVpv, JEVrv, and JEV with cholesterol drastically reduced the infectivity as previously reported, depletion of cholesterol from the particles by treatment with methyl β-cyclodextrin enhanced infectivity. Furthermore, treatment of cells with sphingomyelinase (SMase), which hydrolyzes membrane-bound sphingomyelin to ceramide, drastically enhanced infection with JEVpv and propagation of JEVrv, and these enhancements were inhibited by treatment with an SMase inhibitor or C₆-ceramide. These results suggest that ceramide plays crucial roles in not only entry but also egress processes of JEV, and they should assist in the clarification of JEV propagation and the development of novel therapeutics against diseases caused by infection with flaviviruses.Japanese encephalitis virus (JEV) is a small, enveloped virus belonging to the family Flaviviridae and the genus Flavivirus, which also includes Dengue virus (DENV), West Nile virus (WNV), Yellow fever virus, and Tick-borne encephalitis virus (11). JEV is the most common agent of viral encephalitis, causing approximately 50,000 cases annually, of which 15,000 will die, and up to 50% of survivors are left with severe residual neurological complications. JEV has a single-stranded positive-sense RNA genome of approximately 11 kb, encoding a single large polyprotein, which is cleaved by the host- and virus-encoded proteases into three structural proteins, capsid (C), premembrane (PrM), and envelope (E), and seven nonstructural proteins. The structural proteins are components of viral particles, and the E protein is suggested to interact with a cell surface receptor molecule(s). Although a number of cellular components, including heat shock cognate protein 70 (33), glycosaminoglycans, such as heparin or heparan sulfate (21, 41), and laminin (3), have been shown to participate in JEV infection, the precise mechanisms by which these receptor candidates participate in JEV infection remain largely unclear.In addition to the many studies identifying and characterizing receptor molecules in numerous viruses, data suggesting the involvement of membrane lipids, such as sphingolipids and cholesterol, in viral infection have also been accumulating. Lipid rafts consisting of sphingolipids and cholesterol and distributing to the outer leaflet of the cell membrane have been shown to be involved in the infection of not only many viruses but also several bacteria and parasites (24), in addition to playing roles in various functions such as lipid sorting, protein trafficking (26, 47), cell polarity, and signal transduction (38). With respect to cholesterol itself, various aspects of the life cycle of flaviviruses have been shown to involve this lipid, including the entry of DENV (34), hepatitis C virus (HCV) (16), and WNV (27), the membrane fusion of tick-borne encephalitis virus (40), and the replication of HCV (14, 17), WNV (23), and DENV (35). Recently Lee et al. (20) showed that treatment with cholesterol efficiently impairs both the entry and replication steps of JEV and DENV-2 but enhances infection with the Sindbis virus (22).On the other hand, sphingolipids, including sphingomyelins and glycosphingolipids, are ubiquitous components of eukaryotic cell membrane structures, providing integrity to cellular membranes. Ceramide is one of the intermediates of sphingolipids and plays roles in cell differentiation, regulation of apoptosis and protein secretion, induction of cellular senescence, and other processes (2). Ceramide is generated from the hydrolysis of sphingomyelin by sphingomyelinase (SMase) or from catalysis by serine-palmitoyl-coenzyme A (CoA) transferase and ceramide synthase. Ceramide spontaneously self-associates to form ceramide-enriched microdomains and then to form larger ceramide-enriched membrane platforms which serve as the spatial and temporal organization for cellular signalosomes and for regulation of protein functions (2). The ceramide-enriched platforms have also been used by many pathogens to facilitate entry and infection (2). The acid SMase is activated not only by multiple stimuli, including receptor molecules, gamma irradiation, and some chemicals, but also by infection with some bacteria or viruses (36). Rhinovirus activates the SMase for generation of ceramide and forms ceramide-enriched membrane platforms that serve in the infection of target cells (10). Sindbis virus also activates the SMase and induces apoptosis through a continuous release of ceramide (15). In contrast to these viruses, ceramide inhibits infection with HIV (7) and HCV (48). Ceramide enrichment of the plasma membrane reduces expression of HCV receptor molecules through an ATP-independent internalization and impairs entry of HCV.Pseudotype and recombinant viruses based on the vesicular stomatitis virus (VSV) bearing foreign viral envelope proteins have been shown to be powerful tools for the investigation of viral entry and the development of vaccines. These systems have been used to study infection with viruses that do not propagate readily (31, 43) or that are difficult to handle due to their high-level pathogenicity for humans (42). In addition, the systems allow us to focus on the investigation of entry mechanisms of particular viral envelope proteins by using control viruses harboring an appropriate protein on identical particles.In the present study, we generated pseudotype (JEVpv) and recombinant (JEVrv) VSVs bearing the JEV envelope protein in human cell lines and determined the involvement of sphingolipids, especially ceramide, and cholesterol in infection of human cell lines with JEV. Both JEVpv and JEVrv exhibited infection of target cells via pH- and clathrin-dependent endocytosis. Treatment of cells with cholesterol impaired infection with JEVpv and JEVrv, as previously found in JEV infection (20). In contrast, treatment of cells with SMase drastically enhanced infection with both JEVpv and JEVrv and the production of infectious JEVrv particles. These results indicate that ceramide plays crucial roles in the entry and egress of JEV.     

A Novel Immunochromatographic Test Applied to a Serological Survey of Japanese Encephalitis Virus on Pig Farms in Korea

Among vertebrate species, pigs are a major amplifying host of Japanese encephalitis virus (JEV) and measuring their seroconversion is a reliable indicator of virus activity. Traditionally, the hemagglutination inhibition test has been used for serological testing in pigs; however, it has several limitations and, thus, a more efficient and reliable replacement test is required. In this study, we developed a new immunochromatographic test for detecting antibodies to JEV in pig serum within 15 min. Specifically, the domain III region of the JEV envelope protein was successfully expressed in soluble form and used for developing the immunochromatographic test. The test was then applied to the surveillance of Japanese encephalitis (JE) in Korea. We found that our immunochromatographic test had good sensitivity (84.8%) and specificity (97.7%) when compared with an immunofluorescence assay used as a reference test. During the surveillance of JE in Korea in 2012, the new immunochromatographic test was used to test the sera of 1,926 slaughtered pigs from eight provinces, and 228 pigs (11.8%) were found to be JEV-positive. Based on these results, we also produced an activity map of JEV, which marked the locations of pig farms in Korea that tested positive for the virus. Thus, the immunochromatographic test reported here provides a convenient and effective tool for real-time monitoring of JEV activity in pigs.     

Properties of a Maize Glutathione S-Transferase That Conjugates Coumaric Acid and Other Phenylpropanoids

A glutathione S-transferase (GST) enzyme from corn (Zea mays L. Pioneer hybrid 3906) that is active with p-coumaric acid and other unsaturated phenylpropanoids was purified approximately 97-fold and characterized. The native enzyme appeared to be a monomer with a molecular mass of approximately 30 kD and an apparent isoelectric point at pH 5.2. The enzyme had a pH optimum between 7.5 and 8.0 and apparent Km values of 4.4 and 1.9 mM for reduced glutathione (GSH) and p-coumaric acid, respectively. In addition to p-coumaric acid, the enzyme was also active with o-coumaric acid, m-coumaric acid, trans-cinnamic acid, ferulic acid, and coniferyl alcohol. In addition to GSH, the enzyme could also utilize cysteine as a sulfhydryl source. The enzyme activity measured when GSH and trans-cinnamic acid were used as substrates was enhanced 2.6- and 5.2-fold by the addition of 50 [mu]M p-coumaric acid and 7-hydroxycoumarin, respectively. 1H- and 13C-nuclear magnetic resonance spectroscopic analysis of the conjugate revealed that the enzyme catalyzed the addition of GSH to the olefinic double bond of p-coumaric acid. Based on the high activity and the substrate specificity of this enzyme, it is possible that this enzyme may be involved in the in vivo conjugation of a number of unsaturated phenylpropanoids.     

SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.     

Sampling Design Influences the Observed Dominance of Culex tritaeniorhynchus: Considerations for Future Studies of Japanese Encephalitis Virus Transmission

Mosquito sampling during Japanese encephalitis virus (JEV)-associated studies, particularly in India, has usually been conducted via aspirators or light traps to catch mosquitoes around cattle, which are dead-end hosts for JEV. High numbers of Culex tritaeniorhynchus, relative to other species, have often been caught during these studies. Less frequently, studies have involved sampling outdoor resting mosquitoes. We aimed to compare the relative abundance of mosquito species between these two previously used mosquito sampling methods. From September to December 2013 entomological surveys were undertaken in eight villages in a Japanese encephalitis (JE) endemic area of Bangladesh. Light traps were used to collect active mosquitoes in households, and resting boxes and a Bina Pani Das hop cage were used near oviposition sites to collect resting mosquitoes. Numbers of humans and domestic animals present in households where light traps were set were recorded. In five villages Cx. tritaeniorhynchus was more likely to be selected from light trap samples near hosts than resting collection samples near oviposition sites, according to log odds ratio tests. The opposite was true for Cx. pseudovishnui and Armigeres subalbatus, which can also transmit JEV. Culex tritaeniorhynchus constituted 59% of the mosquitoes sampled from households with cattle, 28% from households without cattle and 17% in resting collections. In contrast Cx. pseudovishnui constituted 5.4% of the sample from households with cattle, 16% from households with no cattle and 27% from resting collections, while Ar. subalbatus constituted 0.15%, 0.38%, and 8.4% of these samples respectively. These observations may be due to differences in timing of biting activity, host preference and host-seeking strategy rather than differences in population density. We suggest that future studies aiming to implicate vector species in transmission of JEV should consider focusing catches around hosts able to transmit JEV.     

Induction of Protective Immunity against Japanese Encephalitis in Mice by Immunization with a Plasmid Encoding Japanese Encephalitis Virus Premembrane and Envelope Genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 μg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 μg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 μg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 μg. The CTLs induced in BALB/c mice immunized twice with 100 μg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Review of Climate,Landscape, and Viral Genetics as Drivers of the Japanese Encephalitis Virus Ecology

The Japanese encephalitis virus (JEV), an arthropod-born Flavivirus, is the major cause of viral encephalitis, responsible for 10,000–15,000 deaths each year, yet is a neglected tropical disease. Since the JEV distribution area has been large and continuously extending toward new Asian and Australasian regions, it is considered an emerging and reemerging pathogen. Despite large effective immunization campaigns, Japanese encephalitis remains a disease of global health concern. JEV zoonotic transmission cycles may be either wild or domestic: the first involves wading birds as wild amplifying hosts; the second involves pigs as the main domestic amplifying hosts. Culex mosquito species, especially Cx. tritaeniorhynchus, are the main competent vectors. Although five JEV genotypes circulate, neither clear-cut genotype-phenotype relationship nor clear variations in genotype fitness to hosts or vectors have been identified. Instead, the molecular epidemiology appears highly dependent on vectors, hosts'' biology, and on a set of environmental factors. At global scale, climate, land cover, and land use, otherwise strongly dependent on human activities, affect the abundance of JEV vectors, and of wild and domestic hosts. Chiefly, the increase of rice-cultivated surface, intensively used by wading birds, and of pig production in Asia has provided a high availability of resources to mosquito vectors, enhancing the JEV maintenance, amplification, and transmission. At fine scale, the characteristics (density, size, spatial arrangement) of three landscape elements (paddy fields, pig farms, human habitations) facilitate or impede movement of vectors, then determine how the JEV interacts with hosts and vectors and ultimately the infection risk to humans. If the JEV is introduced in a favorable landscape, either by live infected animals or by vectors, then the virus can emerge and become a major threat for human health. Multidisciplinary research is essential to shed light on the biological mechanisms involved in the emergence, spread, reemergence, and genotypic changes of JEV.