Tully, Claire M; Chinnakannan, Senthil; Mullarkey, Caitlin E; Ulaszewska, Marta; Ferrara, Francesca; Temperton, Nigel; Gilbert, Sarah C; Lambe, Teresa
Seasonal influenza viruses are a common cause of acute respiratory illness worldwide and generate a significant socioeconomic burden. Influenza viruses mutate rapidly, necessitating annual vaccine reformulation because traditional vaccines do not typically induce broad-spectrum immunity. In addition to seasonal infections, emerging pandemic influenza viruses present a continued threat to global public health. Pandemic influenza viruses have consistently higher attack rates and are typically associated with greater mortality compared with seasonal strains. Ongoing strategies to improve vaccine efficacy typically focus on providing broad-spectrum immunity; although B and T cells can mediate heterosubtypic responses, typical vaccine development will augment either humoral or cellular immunity. However, multipronged approaches that target several Ags may limit the generation of viral escape mutants. There are few vaccine platforms that can deliver multiple Ags and generate robust cellular and humoral immunity. In this article, we describe a novel vaccination strategy, tested preclinically in mice, for the delivery of novel bivalent viral-vectored vaccines. We show this strategy elicits potent T cell responses toward highly conserved internal Ags while simultaneously inducing high levels of Abs toward hemagglutinin. Importantly, these humoral responses generate long-lived plasma cells and generate Abs capable of neutralizing variant hemagglutinin-expressing pseudotyped lentiviruses. Significantly, these novel viral-vectored vaccines induce strong immune responses capable of conferring protection in a stringent influenza A virus challenge. Thus, this vaccination regimen induces lasting efficacy toward influenza. Importantly, the simultaneous delivery of dual Ags may alleviate the selective pressure that is thought to potentiate antigenic diversity in avian influenza viruses. Copyright © 2017 by The American Association of Immunologists, Inc.
Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu
Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. Copyright © 2015 Elsevier Ltd. All rights reserved.
Tripp, Ralph A.; Tompkins, S. Mark
Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformul...
Evans, Michelle V; Dallas, Tad A; Han, Barbara A; Murdock, Courtney C; Drake, John M
Zika is an emerging virus whose rapid spread is of great public health concern. Knowledge about transmission remains incomplete, especially concerning potential transmission in geographic areas in which it has not yet been introduced. To identify unknown vectors of Zika, we developed a data-driven model linking vector species and the Zika virus via vector-virus trait combinations that confer a propensity toward associations in an ecological network connecting flaviviruses and their mosquito vectors. Our model predicts that thirty-five species may be able to transmit the virus, seven of which are found in the continental United States, including Culex quinquefasciatus and Cx. pipiens. We suggest that empirical studies prioritize these species to confirm predictions of vector competence, enabling the correct identification of populations at risk for transmission within the United States. DOI: http://dx.doi.org/10.7554/eLife.22053.001 PMID:28244371
Full Text Available Rice (Oryza sativa L. is cultivated in more than 100 countries and supports nearly half of the world’s population. Developing efficient methods to control rice viruses is thus an urgent necessity because viruses cause serious losses in rice yield. Most rice viruses are transmitted by insect vectors, notably planthoppers and leafhoppers. Viruliferous insect vectors can disperse their viruses over relatively long distances, and eradication of the viruses is very difficult once they become widespread. Exploitation of natural genetic sources of resistance is one of the most effective approaches to protect crops from virus infection; however, only a few naturally occurring rice genes confer resistance against rice viruses. In an effort to improve control, many investigators are using genetic engineering of rice plants as a potential strategy to control viral diseases. Using viral genes to confer pathogen-derived resistance against crops is a well-established procedure, and the expression of various viral gene products has proved to be effective in preventing or reducing infection by various plant viruses since the 1990s. RNA-interference (RNAi, also known as RNA silencing, is one of the most efficient methods to confer resistance against plant viruses on their respective crops. In this article, we review the recent progress, mainly conducted by our research group, in transgenic strategies to confer resistance against tenuiviruses and reoviruses in rice plants. Our findings also illustrate that not all RNAi constructs against viral RNAs are equally effective in preventing virus infection and that it is important to identify the viral Achilles’ heel gene to target for RNAi attack when engineering plants.
Sasaya, Takahide; Nakazono-Nagaoka, Eiko; Saika, Hiroaki; Aoki, Hideyuki; Hiraguri, Akihiro; Netsu, Osamu; Uehara-Ichiki, Tamaki; Onuki, Masatoshi; Toki, Seichi; Saito, Koji; Yatou, Osamu
Rice (Oryza sativa L.) is cultivated in more than 100 countries and supports nearly half of the world's population. Developing efficient methods to control rice viruses is thus an urgent necessity because viruses cause serious losses in rice yield. Most rice viruses are transmitted by insect vectors, notably planthoppers and leafhoppers. Viruliferous insect vectors can disperse their viruses over relatively long distances, and eradication of the viruses is very difficult once they become widespread. Exploitation of natural genetic sources of resistance is one of the most effective approaches to protect crops from virus infection; however, only a few naturally occurring rice genes confer resistance against rice viruses. Many investigators are using genetic engineering of rice plants as a potential strategy to control viral diseases. Using viral genes to confer pathogen-derived resistance against crops is a well-established procedure, and the expression of various viral gene products has proved to be effective in preventing or reducing infection by various plant viruses since the 1990s. RNA interference (RNAi), also known as RNA silencing, is one of the most efficient methods to confer resistance against plant viruses on their respective crops. In this article, we review the recent progress, mainly conducted by our research group, in transgenic strategies to confer resistance against tenuiviruses and reoviruses in rice plants. Our findings also illustrate that not all RNAi constructs against viral RNAs are equally effective in preventing virus infection and that it is important to identify the viral "Achilles' heel" gene to target for RNAi attack when engineering plants.
Kauffman, Elizabeth B; Kramer, Laura D
Zika virus (ZIKV) (Flaviviridae, Flavivirus) has become one of the most medically important mosquito-borne viruses because of its ability to cause microcephaly in utero and Guillain-Barré syndrome in adults. This virus emerged from its sylvatic cycle in Africa to cause an outbreak in Yap, Federated States of Micronesia in 2007, French Polynesia in 2014, and most recently South America in 2015. The rapid expansion of ZIKV in the Americas largely has been due to the biology and behavior of its vector, Aedes aegypti. Other arboviruses transmitted by Ae. aegypti include the 2 flaviviruses dengue virus and yellow fever virus and the alphavirus chikungunya virus, which are also (re)emerging viruses in the Americas. This mosquito vector is highly domesticated, living in close association with humans in urban households. Its eggs are desiccation resistant, and the larvae develop rapidly in subtropical and tropical environments. Climate warming is facilitating range expansion of Ae. aegypti, adding to the threat this mosquito poses to human health, especially in light of the difficulty controlling it. Aedes albopictus, another highly invasive arbovirus vector that has only been implicated in one country (Gabon), is an important vector of ZIKV, but because of its wide geographic distribution may become a more important vector in the future. This article discusses the historical background of ZIKV and the biology and ecology of these 2 vectors. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: email@example.com.
van der Sluijs, Mirjam Tineke Willemijn; de Smit, Abraham J; Moormann, Rob J M
Bluetongue is an economically important disease of ruminants. The causative agent, Bluetongue virus (BTV), is mainly transmitted by insect vectors. This review focuses on vector-free BTV transmission, and its epizootic and economic consequences. Vector-free transmission can either be vertical, from dam to fetus, or horizontal via direct contract. For several BTV-serotypes, vertical (transplacental) transmission has been described, resulting in severe congenital malformations. Transplacental transmission had been mainly associated with live vaccine strains. Yet, the European BTV-8 strain demonstrated a high incidence of transplacental transmission in natural circumstances. The relevance of transplacental transmission for the epizootiology is considered limited, especially in enzootic areas. However, transplacental transmission can have a substantial economic impact due to the loss of progeny. Inactivated vaccines have demonstrated to prevent transplacental transmission. Vector-free horizontal transmission has also been demonstrated. Since direct horizontal transmission requires close contact of animals, it is considered only relevant for within-farm spreading of BTV. The genetic determinants which enable vector-free transmission are present in virus strains circulating in the field. More research into the genetic changes which enable vector-free transmission is essential to better evaluate the risks associated with outbreaks of new BTV serotypes and to design more appropriate control measures.
Nelson Nogueira Reis
Full Text Available Abstract The dengue fever is a major public health problem in the world. In Brazil, in 2015, there were 1,534,932 cases, being 20,320 cases of severe form, and 811 deaths related to this disease. The distribution of Aedes aegypti, the vector, is extensive. Recently, Zika and Chikungunya viruses had arisen, sharing the same vector as dengue and became a huge public health issue. Without specific treatment, it is urgently required as an effective vector control. This article is focused on reviewing vector control strategies, their effectiveness, viability and economical impact. Among all, the Sterile Insect Technique is highlighted as the best option to be adopted in Brazil, once it is largely effectively used in the USA and Mexico for plagues related to agribusiness.
Nelson Nogueira Reis
Full Text Available The dengue fever is a major public health problem in the world. In Brazil, in 2015, there were 1,534,932 cases, being 20,320 cases of severe form, and 811 deaths related to this disease. The distribution of Aedes aegypti, the vector, is extensive. Recently, Zika and Chikungunya viruses had arisen, sharing the same vector as dengue and became a huge public health issue. Without specific treatment, it is urgently required as an effective vector control. This article is focused on reviewing vector control strategies, their effectiveness, viability and economical impact. Among all, the Sterile Insect Technique is highlighted as the best option to be adopted in Brazil, once it is largely effectively used in the USA and Mexico for plagues related to agribusiness.
Wei, Y; Wang, S; Wang, X
Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.
Belliure, B.; Janssen, A.; Maris, P.C.; Peters, D.; Sabelis, M.W.
Plants infected with pathogens often attract the pathogens' vectors, but it is not clear if this is advantageous to the vectors. We therefore quantified the direct and indirect (through the host plant) effects of a pathogen on its vector. A positive direct effect of the plant-pathogenic Tomato
Hoffmann, Bernd; Bauer, Burkhard; Bauer, Christian; Bätza, Hans-Joachim; Beer, Martin; Clausen, Peter-Henning; Geier, Martin; Gethmann, Jörn M.; Kiel, Ellen; Liebisch, Gabriele; Liebisch, Arndt; Mehlhorn, Heinz; Schaub, Günter A.; Werner, Doreen
To identify the vectors of bluetongue virus (BTV) in Germany, we monitored Culicoides spp. biting midges during April 2007–May 2008. Molecular characterization of batches of midges that tested positive for BTV suggests C. obsoletus sensu stricto as a relevant vector of bluetongue disease in central Europe. PMID:19788820
Wu, Qunfeng; Yu, Fulai; Xu, Jinfang; Li, Yang; Chen, Huanchun; Xiao, Shaobo; Fu, Zhen F; Fang, Liurong
Rabies virus has been an ongoing threat to humans and animals. Here, we developed a new strategy to generate a rabies virus vaccine based on a pseudotyped baculovirus. The recombinant baculovirus (BV-RVG/RVG) was pseudotyped with the rabies virus glycoprotein (RVG) and also simultaneously expressed another RVG under the control of the immediate early CMV promoter. In vitro, this RVG-pseudotyped baculovirus vector induced syncytium formation in insect cells and displayed more efficient gene delivery into mammalian cells. Mice immunized with BV-RVG/RVG developed higher levels of virus-neutralizing antibodies, and conferred 100% protection against rabies viral challenge. These data indicate that the RVG-pseudotyped baculovirus BV-RVG/RVG can be used as an alternative strategy to develop a safe and efficacious vaccine against the rabies virus. Copyright © 2014 Elsevier B.V. All rights reserved.
Bouwknegt, C.; Rijn, van P.A.; Schipper, J.M.J.; Holzel, D.R.; Boonstra, J.; Nijhof, A.; Rooij, van E.M.A.; Jongejan, F.
When the first outbreak of bluetongue virus serotype 8 (BTV8) was recorded in North-West Europe in August 2006 and renewed outbreaks occurred in the summer of 2007 and again in 2008, the question was raised how the virus survived the winter. Since most adult Culicoides vector midges are assumed not
Talavera, A; Rodriguez, J M
Vaccinia virus (Vv) is a member of the genus Orthopoxvirus, one of seven genera included in the family Poxviridae. Most of these viruses infect vertebrates (Orthopoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Parapoxvirus), but one genus, Entomopoxvirus, infects insects. It is interesting to note that the Fibroma and Mixoma viruses of the leporipoxvirus genus cause tumors in their hosts (rabbits), these being the only tumorigenic viruses in the family (1,2).
Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders.
Full Text Available Vectors based on adeno-associated virus (AAV are promising tools for gene therapy. The production of strongly toxic vectors, for example for cancer-directed gene transfer, is often unfeasible due to uncontrolled expression of toxic genes in vector-producing cells. Using an approach based on transcriptional repression, we have created novel AAV vectors carrying the genes coding for diphtheria toxin A (DTA and the pro-apoptotic PUMA protein. The DTA vector had a significant toxic effect on a panel of tumor cell lines, and abrogation of protein synthesis could be shown. The PUMA vector had a toxic effect on HeLa and RPMI 8226 cells, and sensitized transduced cells to doxorubicin. To permit targeted gene transfer, we incorporated the DTA gene into a genetically modified AAV-2 capsid previously developed by our group that mediates enhanced transduction of murine breast cancer cells in vitro. This vector had a stronger cytotoxic effect on breast cancer cells than DTA vectors with wildtype AAV capsid or vectors with a random capsid modification. The vector production and application system presented here allows for easy exchange of promotors, transgenes and capsid specificity for certain target cells. It will therefore be of great possible value in a broad range of applications in cytotoxic gene therapy and significantly broadens the spectrum of available tools for AAV-based gene therapy.
Ciota, Alexander T.; Kramer, Laura D.
West Nile virus (WNV; Flavivirus; Flaviviridae) is the cause of the most widespread arthropod-borne viral disease in the world and the largest outbreak of neuroinvasive disease ever observed. Mosquito-borne outbreaks are influenced by intrinsic (e.g., vector and viral genetics, vector and host competence, vector life-history traits) and extrinsic (e.g., temperature, rainfall, human land use) factors that affect virus activity and mosquito biology in complex ways. The concept of vectorial capacity integrates these factors to address interactions of the virus with the arthropod host, leading to a clearer understanding of their complex interrelationships, how they affect transmission of vector-borne disease, and how they impact human health. Vertebrate factors including host competence, population dynamics, and immune status also affect transmission dynamics. The complexity of these interactions are further exacerbated by the fact that not only can divergent hosts differentially alter the virus, but the virus also can affect both vertebrate and invertebrate hosts in ways that significantly alter patterns of virus transmission. This chapter concentrates on selected components of the virus-vector-vertebrate interrelationship, focusing specifically on how interactions between vector, virus, and environment shape the patterns and intensity of WNV transmission. PMID:24351794
Chen, Gong; Pan, Huipeng; Xie, Wen; Wang, Shaoli; Wu, Qingjun; Fang, Yong; Shi, Xiaobin; Zhang, Youjun
Weeds are important in the ecology of field crops, and when crops are harvested, weeds often become the main hosts for plant viruses and their insect vectors. Few studies, however, have examined the relationships between plant viruses, vectors, and weeds. Here, we investigated how infection of the weed Datura stramonium L. by tomato yellow leaf curl virus (TYLCV) affects the host preference and performance of the TYLCV vector, Bemisia tabaci (Gennadius) Q. The results of a choice experiment indicated that B. tabaci Q preferentially settled and oviposited on TYLCV-infected plants rather than on healthy plants. In addition, B. tabaci Q performed better on TYLCV-infected plants than on healthy plants. These results demonstrate that TYLCV is indirectly mutualistic to B. tabaci Q. The mutually beneficial interaction between TYLCV and B. tabaci Q may help explain the concurrent outbreaks of TYLCV and B. tabaci Q in China. PMID:23872717
Brittany L. Dodson
Full Text Available Zika virus is a newly emergent mosquito-borne flavivirus that has caused recent large outbreaks in the new world, leading to dramatic increases in serious disease pathology including Guillain-Barre syndrome, newborn microcephaly, and infant brain damage. Although Aedes mosquitoes are thought to be the primary mosquito species driving infection, the virus has been isolated from dozens of mosquito species, including Culex and Anopheles species, and we lack a thorough understanding of which mosquito species to target for vector control. We exposed Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes to blood meals supplemented with two Zika virus strains. Mosquito bodies, legs, and saliva were collected five, seven, and 14 days post blood meal and tested for infectious virus by plaque assay. Regardless of titer, virus strain, or timepoint, Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes were refractory to Zika virus infection. We conclude that Anopheles gambiae, Anopheles stephensi, and Culex quinquefasciatus mosquitoes likely do not contribute significantly to Zika virus transmission to humans. However, future studies should continue to explore the potential for other novel potential vectors to transmit the virus.
Santiago-Ortiz, Jorge L; Schaffer, David V
Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field. Copyright © 2016 Elsevier B.V. All rights reserved.
Hewitt, F Curtis; Li, Chengwen; Gray, Steven J; Cockrell, Shelley; Washburn, Michael; Samulski, R Jude
Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.
Full Text Available Plant viruses and aphids as their vectors, are limiting factors in the production of healthy seed potato. Potato Virus Y (PVY and Potato Leafroll Virus (PLRV are the two most significant potato viruses in Europe, and seed quality depends directly on the infection level. In order to determine the possibilities for healthy seed potato production in Serbia, aphid flight activities have been monitored for four years in four localities. Over 6400 specimens of aphids have been collected. The number of aphids and vector pressure index varies depending on the localities’ altitude. In localities at altitudes under 1000 m, they were high. The highest index was in locality Kotraža in 2007, when the PVY index exceeded the value of 180, while for PLRV it was 60. At high altitudes, above 1100 m, the number of aphids was low, as was the vector pressure index. The lowest index values were recorded in localities on Mt. Golija at 1300 m a.s.l. where the indexes for both viruses never exceeded value 6. [Projekat Ministarstva nauke Republike Srbije, br. III 46008: Development of integrated management of harmful organisms in plant production in order to overcome resistance and to improve food quality and safety
Osakada, Fumitaka; Callaway, Edward M.
Rabies viruses, negative-strand RNA viruses, infect neurons through axon terminals and spread transsynaptically in a retrograde direction between neurons. Rabies viruses whose glycoprotein (G) gene is deleted from the genome cannot spread across synapses. Complementation of G in trans, however, enables transsynaptic spreading of G-deleted rabies viruses to directly-connected, presynaptic neurons. Recombinant rabies viruses can encode genes of interest for labeling cells, controlling gene expression, and monitoring or manipulating neural activity. Cre-dependent or bridge-protein-mediated transduction and single-cell electroporation via EnvA/TVA or EnvB/TVB system allow cell-type-specific or single-cell-specific targeting. These rabies virus-based approaches permit the linking of connectivity to cell morphology and circuit function for particular cell types or single cells. Here we describe methods for construction of rabies viral vectors, recovery of G-deleted rabies viruses from cDNA, amplification of the viruses, pseudotyping them with EnvA or EnvB, and concentration and titration of the viruses. The entire protocol takes 6–8 weeks. PMID:23887178
Telmadarraiy, Zakkyeh; Chinikar, Sadegh; Vatandoost, Hassan; Faghihi, Faezeh; Hosseini-Chegeni, Asadollah
Background: Ticks are important vectors and reservoirs of Crimean Congo Hemorrhagic Fever (CCHF) virus. Human beings may be infected whenever the normal life cycle of the infected ticks on non-human vertebrate hosts is interrupted by the undesirable presence of humans in the cycle. A total of 26 species of Argasid and Ixodid ticks have been recorded in Iran; including nine Hyalomma, two Rhipicephalus, two Dermacentor, five Haemaphysalis, two Boophilus, one Ixodes and two Argas as well as three Ornithodoros species as blood sucking ectoparasites of livestock and poultries. The present paper reviews tick vectors of CCHF virus in Iran, focusing on the role of ticks in different provinces of Iran using reverse transcription polymerase chain reaction (RT-PCR) assay. Methods: During ten years study, 1054 tick specimens; including two species of Argasidae and 17 species of Ixodidae were examined for their infection to CCHF virus genome. The output of all studies as well as related publications were discussed in the current paper. Results: The results show that Rhipicephalus sanguineus, Hyalomma marginatum, H. anatolicum, H. asiaticum and H. dromedarii were known as the most frequent species which were positive for CCHF virus. Conclusion: The status of ticks which were positive for CCHF virus revealed that unlike the most common idea that Hyalomma species are the most important vectors of CCHF virus, other ticks including Rhipicephalus, Haemaphysalis and Dermacentor can be reservoir of this virus; thus, considering geographical distribution, type of host and environmental conditions, different tick control measurements should be carried out in areas with high incidence of CCHF disease. PMID:26623426
Full Text Available Newcastle disease virus (NDV strain F is a lentogenic vaccine strain used for primary vaccination in day-old chickens against Newcastle disease (ND in India and Southeast Asian countries. Recombinant NDV-F virus and another recombinant NDV harboring the major capsid protein VP2 gene of a very virulent infectious bursal disease virus (IBDV; namely rNDV-F and rNDV-F/VP2, respectively, were generated using the NDV F strain. The rNDV-F/VP2 virus was slightly attenuated, as compared to the rNDV-F virus, as evidenced from the mean death time and intracerebral pathogenicity index analysis. This result indicates that rNDV-F/VP2 behaves as a lentogenic virus and it is stable even after 10 serial passages in embryonated chicken eggs. When chickens were vaccinated with the rNDV F/VP2, it induced both humoral and cell mediated immunity, and was able to confer complete protection against very virulent IBDV challenge and 80% protection against virulent NDV challenge. These results suggest that rNDV-F could be an effective and inherently safe vaccine vector. Here, we demonstrate that a bivalent NDV-IBDV vaccine candidate generated by reverse genetics method is safe, efficacious and cost-effective, which will greatly aid the poultry industry in developing countries.
Terradas, Gerard; McGraw, Elizabeth A
Viruses transmitted by mosquitoes such as dengue, Zika and West Nile cause a threat to global health due to increased geographical range and frequency of outbreaks. The bacterium Wolbachia pipientis may be the solution reducing disease transmission. Though commonly missing in vector species, the bacterium was artificially and stably introduced into Aedes aegypti to assess its potential for biocontrol. When infected with Wolbachia, mosquitoes become refractory to infection by a range of pathogens, including the aforementioned viruses. How the bacterium is conferring this phenotype remains unknown. Here we discuss current hypotheses in the field for the mechanistic basis of pathogen blocking and evaluate the evidence from mosquitoes and related insects. Copyright © 2017 Elsevier Inc. All rights reserved.
Chesnais, Quentin; Couty, Aude; Uzest, Maryline; Brault, Véronique; Ameline, Arnaud
Insect-vectored plant viruses can induce changes in plant phenotypes, thus influencing plant-vector interactions in a way that may promote their dispersal according to their mode of transmission (i.e., circulative vs. noncirculative). This indirect vector manipulation requires host-virus-vector coevolution and would thus be effective solely in very specific plant-virus-vector species associations. Some studies suggest this manipulation may depend on multiple factors relative to various intrinsic characteristics of vectors such as transmission efficiency. In anintegrative study, we tested the effects of infection of the Brassicaceae Camelina sativa with the noncirculative Cauliflower mosaic virus (CaMV) or the circulative Turnip yellows virus (TuYV) on the host-plant colonization of two aphid species differing in their virus transmission efficiency: the polyphagous Myzus persicae, efficient vector of both viruses, and the Brassicaceae specialist Brevicoryne brassicae, poor vector of TuYV and efficient vector of CaMV. Results confirmed the important role of virus mode of transmission as plant-mediated effects of CaMV on the two aphid species induced negative alterations of feeding behavior (i.e., decreased phloem sap ingestion) and performance that were both conducive for virus fitness by promoting dispersion after a rapid acquisition. In addition, virus transmission efficiency may also play a role in vector manipulation by viruses as only the responses of the efficient vector to plant-mediated effects of TuYV, that is, enhanced feeding behavior and performances, were favorable to their acquisition and further dispersal. Altogether, this work demonstrated that vector transmission efficiency also has to be considered when studying the mechanisms underlying vector manipulation by viruses. Our results also reinforce the idea that vector manipulation requires coevolution between plant, virus and vector. © 2017 Institute of Zoology, Chinese Academy of Sciences.
Ramsauer, Katrin; Tangy, Frédéric
In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail firstname.lastname@example.org.
Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.
Bastone, P.; Romen, F.; Liu, W.; Wirtz, R.; Koch, U.; Josephson, N.; Langbein, S.; Löchelt, M.
As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are
Bragard, C.; Caciagli, P.; Lemaire, O.; Lopez-Moya, J.J.; MacFarlane, S.; Peters, D.; Susi, P.; Torrance, L.
Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus–vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers,
Dmitry M Shayakhmetov; Carlson, Cheryl A.; Stecher, Hartmut; Li, Qiliang; Stamatoyannopoulos, George; Lieber, André
To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, ΔAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The ΔAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity ΔAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenov...
van Mölken, Tamara; Caluwe, Hannie de; Hordijk, Cornelis A.
viruses and non-vectoring herbivores. We investigated the effects of virus infection on subsequent infestation by a non-vectoring herbivore in a natural genotype of Trifolium repens (white clover). We tested whether infection with White clover mosaic virus (WClMV) alters (1) the effects of fungus gnat...
Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long
Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.
Scott, Thomas W; Morrison, Amy C
Accounting for variation in mosquito vector populations will improve dengue surveillance and prevention. Because Aedes aegypti, the principle dengue virus (DENV) vector, transmit the virus with remarkable efficiency, entomological thresholds are especially low. Assessing risk of human infection based on immature mosquito indices has proven difficult. Greater emphasis should be placed on relative abundance of adult vectors in relation to human serotype-specific herd immunity, introduction of unique viruses, mosquito-human contact and weather. The most appropriate spatial scale for assessing entomological risk is the individual household. The scale for measuring DENV transmission risk has yet to be determined but is clearly larger than the household and likely to exceed several city blocks. Because households are expected to be a primary site for human DENV infection, intradomicile vector control strategies should be a priority, especially when the force of transmission is high. The most effective intervention strategy will combine vector control with vaccine delivery for rapid and sustained disease prevention.
Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung
Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.
Ayşegül Akbay Yarpuzlu
Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9
De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.
ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411
Lacorte, C.C.; Ribeiro, S.G.; Lohuis, H.; Goldbach, R.W.; Prins, M.W.
Virus-based expression vectors are important tools for high-level production of foreign proteins and for gene function analysis through virus induced gene silencing. To exploit further their advantages as fast, high yield replicons, a set of vectors was produced by converting and adapting Potato
de Wit, Emmie; Marzi, Andrea; Bushmaker, Trenton; Brining, Doug; Scott, Dana; Richt, Juergen A; Geisbert, Thomas W; Feldmann, Heinz
The ongoing Ebola outbreak in West Africa has resulted in fast-track development of vaccine candidates. We tested a vesicular stomatitis virus vector expressing Ebola virus glycoprotein for safety in pigs. Inoculation did not cause disease and vaccine virus shedding was minimal, which indicated that the vaccine virus does not pose a risk of dissemination in pigs.
Spaete, R R; Frenkel, N
We have employed repeat units of herpes simplex virus (HSV) defective genomes to derive a cloning-amplifying vector (amplicon) that can replicate in eucaryotic cells in the presence of standard HSV helper virus. The design of the HSV amplicon system is based on the previous observation that cotransfection of cells with helper virus DNA and seed monomeric repeat units of HSV defective genomes results in the regeneration of concatemeric defective genomes composed of multiple reiterations of the seed repeats. Cotransfection of cells with helper virus DNA and chimeric repeat units containing bacterial plasmid pKC7 DNA resulted in the generation of defective genomes composed of reiterations of the seed HSV-pKC7 repeats. These chimeric defective genomes were packaged into virus particles and could be propagated in virus stocks, with the most enriched passages containing more than 90% chimeric defective genomes. Furthermore, monomeric chimeric repeat units could be transferred back and forth between bacteria and eucaryotic cells. A derivative vector constructed so as to contain several unique restriction enzyme sites could be potentially employed in the introduction of additional viral or eucaryotic DNA sequences into eucaryotic cells.
Dawson, William O; Folimonova, Svetlana Y
Virus-based expression vectors are commonplace tools for the production of proteins or the induction of RNA silencing in herbaceous plants. This review considers a completely different set of uses for viral vectors in perennial fruit and nut crops, which can be productive for periods of up to 100 years. Viral vectors could be used in the field to modify existing plants. Furthermore, with continually emerging pathogens and pests, viral vectors could express genes to protect the plants or even to treat plants after they become infected. As technologies develop during the life span of these crops, viral vectors can be used for adding new genes as an alternative to pushing up the crop and replanting with transgenic plants. Another value of virus-based vectors is that they add nothing permanently to the environment. This requires that effective and stable viral vectors be developed for specific crops from endemic viruses. Studies using viruses from perennial hosts suggest that these objectives could be accomplished.
Sarver, N; Gruss, P; Law, M F; Khoury, G; Howley, P M
A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin. Images PMID:6100967
Tassi, Aline Daniele; Garita-Salazar, Laura Cristina; Amorim, Lilian; Novelli, Valdenice Moreira; Freitas-Astúa, Juliana; Childers, Carl C; Kitajima, Elliot W
Citrus leprosis has been one of the most destructive diseases of citrus in the Americas. In the last decade important progress has been achieved such as the complete genome sequencing of its main causal agent, Citrus leprosis virus C (CiLV-C), belonging to a new genus Cilevirus. It is transmitted by Brevipalpus yothersi Baker (Acari: Tenuipalpidae), and is characterized by the localized symptoms it induces on the leaves, fruits and stems. It occurs in the American continents from Mexico to Argentina. The virus was until recently considered restricted to Citrus spp. However, it was found naturally infecting other plants species as Swinglea glutinosa Merrill and Commelina benghalensis L., and has been experimentally transmitted by B. yothersi to a large number of plant species. Despite these advances little is known about the virus-vector relationship that is a key to understanding the epidemiology of the disease. Some components of the CiLV-C/B. yothersi relationship were determined using the common bean (Phaseolus vulgaris L. cv. 'IAC Una') as a test plant. They included: (a) the virus acquisition access period was 4 h; (b) the virus inoculation access period was 2 h; (c) the latent period between acquisition and inoculation was 7 h; (d) the period of retention of the virus by a single viruliferous mite was at least 12 days; (d) the percentage of viruliferous individuals from mite colonies on infected tissues ranged from 25 to 60%. The experiments confirmed previous data that all developmental stages of B. yothersi (larva, protonymph and deutonymph, adult female and male) were able to transmit CiLV-C and that transovarial transmission of the virus did not occur. CiLV-C can be acquired from lesions on leaves, fruits and stems by B. yothersi. Based on the distribution of lesions produced by single viruliferous B. yothersi on bean leaves, it is concluded that they tend to feed in restricted areas, usually near the veins. The short latent and transmission periods
Manrique-Saide, Pablo; Che-Mendoza, Azael; Barrera-Perez, Mario; Guillermo-May, Guillermo; Herrera-Bojorquez, Josue; Dzul-Manzanilla, Felipe; Gutierrez-Castro, Cipriano; Lenhart, Audrey; Vazquez-Prokopec, Gonzalo; Sommerfeld, Johannes; McCall, Philip J; Kroeger, Axel; Arredondo-Jimenez, Juan I
Dengue prevention efforts rely on control of virus vectors. We investigated use of insecticide-treated screens permanently affixed to windows and doors in Mexico and found that the screens significantly reduced infestations of Aedes aegypti mosquitoes in treated houses. Our findings demonstrate the value of this method for dengue virus vector control.
Full Text Available Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed.
Kliot, Adi; Ghanim, Murad
Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed.
Adaeze O Izuogu
Full Text Available Tick-borne flaviviruses (TBFVs, including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1-30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1 or the type I IFN receptor (IFNAR1 by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic
Manuella van Munster
Full Text Available Drought is a major threat to crop production worldwide and is accentuated by global warming. Plant responses to this abiotic stress involve physiological changes overlapping, at least partially, the defense pathways elicited both by viruses and their herbivore vectors. Recently, a number of theoretical and empirical studies anticipated the influence of climate changes on vector-borne viruses of plants and animals, mainly addressing the effects on the virus itself or on the vector population dynamics, and inferring possible consequences on virus transmission. Here, we directly assess the effect of a severe water deficit on the efficiency of aphid-transmission of the Cauliflower mosaic virus (CaMV or the Turnip mosaic virus (TuMV. For both viruses, our results demonstrate that the rate of vector-transmission is significantly increased from water-deprived source plants: CaMV transmission reproducibly increased by 34% and that of TuMV by 100%. In both cases, the enhanced transmission rate could not be explained by a higher virus accumulation, suggesting a more complex drought-induced process that remains to be elucidated. The evidence that infected plants subjected to drought are much better virus sources for insect vectors may have extensive consequences for viral epidemiology, and should be investigated in a wide range of plant-virus-vector systems.
Bragard, C; Caciagli, P; Lemaire, O; Lopez-Moya, J J; MacFarlane, S; Peters, D; Susi, P; Torrance, L
Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.
Full Text Available Abstract Background The risk posed to the United Kingdom by West Nile virus (WNV has previously been considered low, due to the absence or scarcity of the main Culex sp. bridge vectors. The mosquito Culex modestus is widespread in southern Europe, where it acts as the principle bridge vector of WNV. This species was not previously thought to be present in the United Kingdom. Findings Mosquito larval surveys carried out in 2010 identified substantial populations of Cx. modestus at two sites in marshland in southeast England. Host-seeking-adult traps placed at a third site indicate that the relative seasonal abundance of Cx. modestus peaks in early August. DNA barcoding of these specimens from the United Kingdom and material from southern France confirmed the morphological identification. Conclusions Cx. modestus appears to be established in the North Kent Marshes, possibly as the result of a recent introduction. The addition of this species to the United Kingdom's mosquito fauna may increase the risk posed to the United Kingdom by WNV.
Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin
Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857
Chen, Yong; Chen, Qian; Li, Manman; Mao, Qianzhuo; Chen, Hongyan; Wu, Wei; Jia, Dongsheng; Wei, Taiyun
Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.
Mei, Yu; Whitham, Steven A
Virus-induced gene silencing (VIGS) is a powerful technology for rapidly and transiently knocking down the expression of plant genes to study their functions. A VIGS vector for maize derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus, was recently developed. A cloning site created near the 3' end of the FoMV genome enables insertion of 200-400 nucleotide fragments of maize genes targeted for silencing. The recombinant FoMV clones are inoculated into leaves of maize seedlings by biolistic particle delivery, and silencing is typically observed within 2 weeks after inoculation. This chapter provides a protocol for constructing FoMV VIGS clones and inoculating them into maize seedlings.
Nasar, Farooq; Haddow, Andrew D; Tesh, Robert B; Weaver, Scott C
Most alphaviruses are arthropod-borne and utilize mosquitoes as vectors for transmission to susceptible vertebrate hosts. This ability to infect both mosquitoes and vertebrates is essential for maintenance of most alphaviruses in nature. A recently characterized alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani s.I. is unable to replicate in vertebrate cell lines. The EILV host range restriction occurs at both attachment/entry as well as genomic RNA replication levels. Here we investigated the mosquito vector range of EILV in species encompassing three genera that are responsible for maintenance of other alphaviruses in nature. Susceptibility studies were performed in four mosquito species: Aedes albopictus, A. aegypti, Anopheles gambiae, and Culex quinquefasciatus via intrathoracic and oral routes utilizing EILV and EILV expressing red fluorescent protein (-eRFP) clones. EILV-eRFP was injected at 10(7) PFU/mL to visualize replication in various mosquito organs at 7 days post-infection. Mosquitoes were also injected with EILV at 10(4)-10(1) PFU/mosquito and virus replication was measured via plaque assays at day 7 post-infection. Lastly, mosquitoes were provided bloodmeals containing EILV-eRFP at doses of 10(9), 10(7), 10(5) PFU/mL, and infection and dissemination rates were determined at 14 days post-infection. All four species were susceptible via the intrathoracic route; however, replication was 10-100 fold less than typical for most alphaviruses, and infection was limited to midgut-associated muscle tissue and salivary glands. A. albopictus was refractory to oral infection, while A. gambiae and C. quinquefasciatus were susceptible only at 10(9) PFU/mL dose. In contrast, A. aegypti was susceptible at both 10(9) and 10(7) PFU/mL doses, with body infection rates of 78% and 63%, and dissemination rates of 26% and 8%, respectively. The exclusion of vertebrates in its maintenance cycle may have facilitated the adaptation of EILV to a single
Koff, W. C.; Parks, C. L.; Berkhout, B.; Ackland, J.; Noble, S.; Gust, I. D.
At the International AIDS Society Conference oil Pathogenesis, Treatment and Prevention held in Sydney, Australia, in July 2007, the International AIDS Vaccine Initiative (IAVI) convened a satellite symposium entitled 'Accelerating the Development of Replicating Viral Vectors for AIDS Vaccines.' Its
Dietzgen, Ralf G; Mann, Krin S; Johnson, Karyn N
Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus-insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors.
Full Text Available Duck Tembusu virus (DTMUV is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV was examined as a candidate vaccine vector to deliver the envelope (E of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC of C-KCE (vBAC-C-KCE. The envelope (E gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs. Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.
Pérez Jiménez, Eva; Larraga, Vicente; Esteban, Mariano
Vectores recombinantes basados en el virus vaccinia modificado de Ankara (MVA) como vacunas contra la leishmaniasis. Los vectores de la invención contienen secuencias codificantes de la proteína LACK, preferentemente insertadas en el locus de hemaglutinina del virus y bajo el control de un promotor que permite su expresión a lo largo del ciclo de infección del virus. Son vectores seguros, estables, que dan lugar a una potente respuesta inmune que confiere protección frente a la leishmaniasis,...
Full Text Available The interaction between plant viruses and non-vector arthropod herbivores is poorly understood. However, there is accumulating evidence that plant viruses can impact fitness of non-vector herbivores. In this study, we used oligonucleotide microarrays, phytohormone, and total free amino acid analyses to characterize the molecular mechanisms underlying the interaction between Tomato spotted wilt virus (TSWV and a non-vector arthropod, twospotted spider mite (Tetranychusurticae, on tomato plants, Solanumlycopersicum. Twospotted spider mites showed increased preference for and fecundity on TSWV-infected plants compared to mock-inoculated plants. Transcriptome profiles of TSWV-infected plants indicated significant up-regulation of salicylic acid (SA-related genes, but no apparent down-regulation of jasmonic acid (JA-related genes which could potentially confer induced resistance against TSM. This suggests that there was no antagonistic crosstalk between the signaling pathways to influence the interaction between TSWV and spider mites. In fact, SA- and JA-related genes were up-regulated when plants were challenged with both TSWV and the herbivore. TSWV infection resulted in down-regulation of cell wall-related genes and photosynthesis-associated genes, which may contribute to host plant susceptibility. There was a three-fold increase in total free amino acid content in virus-infected plants compared to mock-inoculated plants. Total free amino acid content is critical for arthropod nutrition and may, in part, explain the apparent positive indirect effect of TSWV on spider mites. Taken together, these data suggest that the mechanism(s of increased host suitability of TSWV-infected plants to non-vector herbivores is complex and likely involves several plant biochemical processes.
Nancy J Sullivan
Full Text Available Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd encoding the Ebola glycoprotein (GP and nucleoprotein (NP has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine.To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10 particles, two logs lower than that used previously.Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate.
Shayakhmetov, Dmitry M; Carlson, Cheryl A; Stecher, Hartmut; Li, Qiliang; Stamatoyannopoulos, George; Lieber, André
To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, DeltaAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The DeltaAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity DeltaAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human gamma-globin gene and the HS2 and HS3 elements of the beta-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with DeltaAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.
Ding, Xin Shun; Mannas, Stephen W; Bishop, Bethany A; Rao, Xiaolan; Lecoultre, Mitchell; Kwon, Soonil; Nelson, Richard S
Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 ( HSP70-1 ) inserts in Nicotiana benthamiana and maize ( Zea mays ). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70 , silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. © 2018 American Society of Plant Biologists. All Rights Reserved.
Full Text Available Newly emerging influenza viruses continue to threaten public health. A rapid determination of the host range of newly discovered influenza viruses would assist in early assessment of their risk. Here, we attempted to predict the host of influenza viruses using the Support Vector Machine (SVM classifier based on the word vector, a new representation and feature extraction method for biological sequences. The results show that the length of the word within the word vector, the sequence type (DNA or protein and the species from which the sequences were derived for generating the word vector all influence the performance of models in predicting the host of influenza viruses. In nearly all cases, the models built on the surface proteins hemagglutinin (HA and neuraminidase (NA (or their genes produced better results than internal influenza proteins (or their genes. The best performance was achieved when the model was built on the HA gene based on word vectors (words of three-letters long generated from DNA sequences of the influenza virus. This results in accuracies of 99.7% for avian, 96.9% for human and 90.6% for swine influenza viruses. Compared to the method of sequence homology best-hit searches using the Basic Local Alignment Search Tool (BLAST, the word vector-based models still need further improvements in predicting the host of influenza A viruses.
Virus-induced gene silencing (VIGS) is a widely used tool for gene function studies in many plant species, though its use in monocots has been limited. Using a Brome mosaic virus (BMV) vector designed to silence the maize phytoene desaturase gene, a genetically diverse set of maize inbred lines was ...
Chattopadhyay, Anasuya; Rose, John K
Replication-defective vaccine vectors based on vesicular stomatitis virus (VSV) lacking its envelope glycoprotein gene (G) are highly effective in animal models. However, such ΔG vectors are difficult to grow because they require complementation with the VSV G protein. In addition, the complementing G protein induces neutralizing antibodies in animals and thus limits multiple vector applications. In the process of generating an experimental Nipah virus (a paramyxovirus) vaccine, we generated two defective VSVΔG vectors, each expressing one of the two Nipah virus (NiV) glycoproteins (G and F) that are both required for virus entry to host cells. These replication-defective VSV vectors were effective at generating NiV neutralizing antibody in mice. Most interestingly, we found that these two defective viruses could be grown together and passaged in tissue culture cells in the absence of VSV G complementation. This mixture of complementing defective viruses was also highly effective at generating NiV neutralizing antibody in animals. This novel approach to growing and producing a vaccine from two defective viruses could be generally applicable to vaccine production for other paramyxoviruses or for other viruses where the expression of at least two different proteins is required for viral entry. Such an approach minimizes biosafety concerns that could apply to single, replication-competent VSV recombinants expressing all proteins required for infection.
Burgers, Wendy A; Ginbot, Zekarias; Shen, Yen-Ju; Chege, Gerald K; Soares, Andreia P; Müller, Tracey L; Bunjun, Rubina; Kiravu, Agano; Munyanduki, Henry; Douglass, Nicola; Williamson, Anna-Lise
Poxvirus vectors represent promising human immunodeficiency virus (HIV) vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant capripoxvirus vector, lumpy skin disease virus (LSDV), in combination with modified vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrated that the combination regimen was immunogenic in rhesus macaques, inducing high-magnitude, broad and balanced CD4(+) and CD8(+) T-cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector. © 2014 The Authors.
Schuster, Benjamin S; Kim, Anthony J; Kays, Joshua C; Kanzawa, Mia M; Guggino, William B; Boyle, Michael P; Rowe, Steven M; Muzyczka, Nicholas; Suk, Jung Soo; Hanes, Justin
.... We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials...
Liu, Zhuanzhuan; Zhou, Tengfei; Lai, Zetian; Zhang, Zhenhong; Jia, Zhirong; Zhou, Guofa; Williams, Tricia; Xu, Jiabao; Gu, Jinbao; Zhou, Xiaohong; Lin, Lifeng; Yan, Guiyun; Chen, Xiao-Guang
In China, the prevention and control of Zika virus disease has been a public health threat since the first imported case was reported in February 2016. To determine the vector competence of potential vector mosquito species, we experimentally infected Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes and determined infection rates, dissemination rates, and transmission rates. We found the highest vector competence for the imported Zika virus in Ae. aegypti mosquitoes, some susceptibility of Ae. albopictus mosquitoes, but no transmission ability for Cx. quinquefasciatus mosquitoes. Considering that, in China, Ae. albopictus mosquitoes are widely distributed but Ae. aegypti mosquito distribution is limited, Ae. albopictus mosquitoes are a potential primary vector for Zika virus and should be targeted in vector control strategies.
Liu, Zhuanzhuan; Zhou, Tengfei; Lai, Zetian; Zhang, Zhenhong; Jia, Zhirong; Zhou, Guofa; Williams, Tricia; Xu, Jiabao; Gu, Jinbao; Zhou, Xiaohong; Lin, Lifeng; Yan, Guiyun
In China, the prevention and control of Zika virus disease has been a public health threat since the first imported case was reported in February 2016. To determine the vector competence of potential vector mosquito species, we experimentally infected Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes and determined infection rates, dissemination rates, and transmission rates. We found the highest vector competence for the imported Zika virus in Ae. aegypti mosquitoes, some susceptibility of Ae. albopictus mosquitoes, but no transmission ability for Cx. quinquefasciatus mosquitoes. Considering that, in China, Ae. albopictus mosquitoes are widely distributed but Ae. aegypti mosquito distribution is limited, Ae. albopictus mosquitoes are a potential primary vector for Zika virus and should be targeted in vector control strategies. PMID:28430562
Roy, Avijit; Hartung, John S; Schneider, William L; Shao, Jonathan; Leon, Guillermo; Melzer, Michael J; Beard, Jennifer J; Otero-Colina, Gabriel; Bauchan, Gary R; Ochoa, Ronald; Brlansky, Ronald H
Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.
Schön, Christian; Biel, Martin; Michalakis, Stylianos
Adeno-associated virus (AAV) vectors are the most widely used vehicle systems for neuronal gene transfer. This popularity is based on the non-pathogenic nature of AAVs and their versatility making them a multifunctional vector system for basic research and clinical applications. AAVs are successfully applied in clinical and pre-clinical gene therapy studies for inherited retinal disorders. Their excellent transduction profile and efficiency also boosted the use of AAV vectors in basic research. The AAV vector system can be easily modified and adjusted at multiple levels to allow for optimized and specific gene expression in target cells. Here, we will provide an overview on the AAV vector system and its applications focusing on gene transfer into retinal cells. Furthermore, we will outline and discuss strategies for the optimization of AAV gene transfer by modifications to the AAV vector expression cassette, the AAV capsid or the routes of vector administration. Copyright © 2015 Elsevier B.V. All rights reserved.
Luan, Jun-Bo; Yao, Dan-Mei; Zhang, Tong; Walling, Linda L; Yang, Mei; Wang, Yu-Jun; Liu, Shu-Sheng
Vectors often perform better on plants infected with pathogens, and this promotes the spread of pathogens. However, few studies have examined how plant defensive compounds mediate such mutualistic relationships. Although tobacco plants are relatively poor host plants for the whitefly Bemisia tabaci, tobacco's suitability to the whitefly was substantially increased when infected by the begomovirus Tomato yellow leaf curl China virus. The change in suitability was associated with induced terpenoid synthesis in whitefly-infested plants and repressed terpenoid synthesis in virus-infected plants. Elevation of terpenoid levels via exogenous stem applications reduced the performance of whiteflies. In contrast, suppression of terpenoid synthesis via gene silencing improved whitefly fitness. By integrating genomics, transcriptomics and metabolomics, this study demonstrated that virus infection depleted the terpenoid-mediated plant defence against whiteflies, thereby favouring vector-virus mutualism. These data suggest that plant terpenoids play a key role in shaping vector-pathogen relationships. © 2012 Blackwell Publishing Ltd/CNRS.
Full Text Available Few other diseases exert such a huge toll of suffering as influenza. We report here that intranasal (i.n. administration of E1/E3-defective (ΔE1E3 adenovirus serotype 5 (Ad5 particles rapidly induced an anti-influenza state as a means of prophylactic therapy which persisted for several weeks in mice. By encoding an influenza virus (IFV hemagglutinin (HA HA1 domain, an Ad5-HA1 vector conferred rapid protection as a prophylactic drug followed by elicitation of sustained protective immunity as a vaccine for inducing seamless protection against influenza as a drug-vaccine duo (DVD in a single package. Since Ad5 particles induce a complex web of host responses, which could arrest influenza by activating a specific arm of innate immunity to impede IFV growth in the airway, it is conceivable that this multi-pronged influenza DVD may escape the fate of drug resistance that impairs the current influenza drugs.
Kurth, Elizabeth G; Peremyslov, Valera V; Prokhnevsky, Alexey I; Kasschau, Kristin D; Miller, Marilyn; Carrington, James C; Dolja, Valerian V
The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.
Betancourt, Dillon; de Queiroz, Nina M G P; Xia, Tianli; Ahn, Jeonghyun; Barber, Glen N
Zika virus (ZIKV) has become a serious public health concern because of its link to brain damage in developing human fetuses. Recombinant vesicular stomatitis virus (rVSV) was shown to be a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. In this study, we generated rVSVs (wild-type and attenuated VSV with mutated matrix protein [VSVm] versions) that express either the full length ZIKV envelope protein (ZENV) alone or include the ZENV precursor to the membrane protein upstream of the envelope protein, and our rVSV-ZIKV constructs showed efficient immunogenicity in murine models. We also demonstrated maternal protective immunity in challenged newborn mice born to female mice vaccinated with VSVm-ZENV containing the transmembrane domain. Our data indicate that rVSVm may be a suitable strategy for the design of effective vaccines against ZIKV. Copyright © 2017 by The American Association of Immunologists, Inc.
Chaskopoulou, Alexandra; L'Ambert, Gregory; Petric, Dusan; Bellini, Romeo; Zgomba, Marija; Groen, Thomas A; Marrama, Laurence; Bicout, Dominique J
West Nile virus (WNV) represents a serious burden to human and animal health because of its capacity to cause unforeseen and large epidemics. Until 2004, only lineage 1 and 3 WNV strains had been found in Europe. Lineage 2 strains were initially isolated in 2004 (Hungary) and in 2008 (Austria) and for the first time caused a major WNV epidemic in 2010 in Greece with 262 clinical human cases and 35 fatalities. Since then, WNV lineage 2 outbreaks have been reported in several European countries including Italy, Serbia and Greece. Understanding the interaction of ecological factors that affect WNV transmission is crucial for preventing or decreasing the impact of future epidemics. The synchronous co-occurrence of competent mosquito vectors, virus, bird reservoir hosts, and susceptible humans is necessary for the initiation and propagation of an epidemic. Weather is the key abiotic factor influencing the life-cycles of the mosquito vector, the virus, the reservoir hosts and the interactions between them. The purpose of this paper is to review and compare mosquito population dynamics, and weather conditions, in three ecologically different contexts (urban/semi-urban, rural/agricultural, natural) across four European countries (Italy, France, Serbia, Greece) with a history of WNV outbreaks. Local control strategies will be described as well. Improving our understanding of WNV ecology is a prerequisite step for appraising and optimizing vector control strategies in Europe with the ultimate goal to minimize the probability of WNV infection.
Blagrove, Marcus S C; Caminade, Cyril; Waldmann, Elisabeth; Sutton, Elizabeth R; Wardeh, Maya; Baylis, Matthew
Mosquito-borne viruses have been estimated to cause over 100 million cases of human disease annually. Many methodologies have been developed to help identify areas most at risk from transmission of these viruses. However, generally, these methodologies focus predominantly on the effects of climate on either the vectors or the pathogens they spread, and do not consider the dynamic interaction between the optimal conditions for both vector and virus. Here, we use a new approach that considers the complex interplay between the optimal temperature for virus transmission, and the optimal climate for the mosquito vectors. Using published geolocated data we identified temperature and rainfall ranges in which a number of mosquito vectors have been observed to co-occur with West Nile virus, dengue virus or chikungunya virus. We then investigated whether the optimal climate for co-occurrence of vector and virus varies between "warmer" and "cooler" adapted vectors for the same virus. We found that different mosquito vectors co-occur with the same virus at different temperatures, despite significant overlap in vector temperature ranges. Specifically, we found that co-occurrence correlates with the optimal climatic conditions for the respective vector; cooler-adapted mosquitoes tend to co-occur with the same virus in cooler conditions than their warmer-adapted counterparts. We conclude that mosquitoes appear to be most able to transmit virus in the mosquitoes' optimal climate range, and hypothesise that this may be due to proportionally over-extended vector longevity, and other increased fitness attributes, within this optimal range. These results suggest that the threat posed by vector-competent mosquito species indigenous to temperate regions may have been underestimated, whilst the threat arising from invasive tropical vectors moving to cooler temperate regions may be overestimated.
Viruses are likely to be the most dangerous parasites of living organisms because of their widespread occurrence, possible deleterious effects on their hosts and high rates of evolution. Virus host-to-host transmission is a critical step in the virus life cycle, because it enables survival in a given environment and efficient dissemination. As hosts of plant viruses are not mobile, these pathogens have adopted diverse transmission strategies involving various vector organisms, mainly arthropods, nematodes, fungi and protists. In nature, plants are often infected with more than one virus at a time, thereby creating potential sources for vectors to acquire and transmit simultaneously two or more viruses. Simultaneous transmission can result in multiple infections of new host plants, which become subsequent potential sources of the viruses, thus enhancing the spread of the diseases caused by these pathogens. Moreover, it can contribute to the maintenance of viral genetic diversity in the host communities. However, despite its possible significance, the problem of the simultaneous transmission of plant viruses by vectors has not been investigated in detail. In this review, the current knowledge on multiple viral transmissions by aphids, whiteflies, leafhoppers, planthoppers, nematodes and fungi is outlined. © 2013 BSPP AND JOHN WILEY & SONS LTD.
Tomato spotted wilt virus (TSWV), member of the genus Tospovirus within the family Bunyaviridae, ranks among the top ten of economically most important plant viruses. Tospoviruses cause significant yield losses in agricultural crops such as tomato,
Navarro; Oudrhiri; Fabrega; Lehn
Although most research in the field of somatic gene therapy has investigated the use of recombinant viruses for transferring genes into somatic target cells, various methods for nonviral gene delivery have also been proposed. Both types of gene delivery systems have advantages and drawbacks. Schematically, viral vectors are particularly efficient for gene delivery, whereas nonviral systems are free of the difficulties associated with the use of recombinant viruses but need to be further optimized to reach their full potential. In order to bridge the gap between viral vectors and synthetic reagents, we discuss here some specific features of the viral vector systems of today that could advantageously be taken into account for the design of improved nonviral gene delivery systems. Indeed, although nonviral systems differ fundamentally from viral systems, one possible approach towards enhanced artificial reagents aims at developing 'artificial viruses' that mimic the highly efficient processes of viral infection.
Hefferon, Kathleen Laura
Transgenic plants present enormous potential as a cost-effective and safe platform for large-scale production of vaccines and other therapeutic proteins. A number of different technologies are under development for the production of pharmaceutical proteins from plant tissues. One method used to express high levels of protein in plants involves the employment of plant virus expression vectors. Plant virus vectors have been designed to carry vaccine epitopes as well as full therapeutic proteins such as monoclonal antibodies in plant tissue both safely and effectively. Biopharmaceuticals such as these offer enormous potential on many levels, from providing relief to those who have little access to modern medicine, to playing an active role in the battle against cancer. This review describes the current design and status of plant virus expression vectors used as production platforms for biopharmaceutical proteins. Copyright © 2012 Elsevier Inc. All rights reserved.
Bialosuknia, Sean M.; Zink, Steven D.; Brecher, Matthew; Ehrbar, Dylan J.; Morrissette, Madeline N.; Kramer, Laura D.
In the Western Hemisphere, Zika virus is thought to be transmitted primarily by Aedes aegypti mosquitoes. To determine the extent to which Ae. albopictus mosquitoes from the United States are capable of transmitting Zika virus and the influence of virus dose, virus strain, and mosquito species on vector competence, we evaluated multiple doses of representative Zika virus strains in Ae. aegypti and Ae. albopictus mosquitoes. Virus preparation (fresh vs. frozen) significantly affected virus infectivity in mosquitoes. We calculated 50% infectious doses to be 6.1–7.5 log10 PFU/mL; minimum infective dose was 4.2 log10 PFU/mL. Ae. albopictus mosquitoes were more susceptible to infection than Ae. aegypti mosquitoes, but transmission efficiency was higher for Ae. aegypti mosquitoes, indicating a transmission barrier in Ae. albopictus mosquitoes. Results suggest that, although Zika virus transmission is relatively inefficient overall and dependent on virus strain and mosquito species, Ae. albopictus mosquitoes could become major vectors in the Americas. PMID:28430564
Full Text Available X-linked severe combined immunodeficiency (SCID-X1 is an inherited genetic immunodeficiency associated with mutations in the common cytokine receptor γ chain (γc gene, and characterized by a complete defect of T and natural killer (NK cells. Gene therapy for SCID-X1 using conventional retroviral (RV vectors carrying the γc gene results in the successful reconstitution of T cell immunity. However, the high incidence of vector-mediated T cell leukemia, caused by vector insertion near or within cancer-related genes has been a serious problem. In this study, we established a gene therapy model of mouse SCID-X1 using a modified foamy virus (FV vector expressing human γc. Analysis of vector integration in a human T cell line demonstrated that the FV vector integration sites were significantly less likely to be located within or near transcriptional start sites than RV vector integration sites. To evaluate the therapeutic efficacy, bone marrow cells from γc-knockout (γc-KO mice were infected with the FV vector and transplanted into γc-KO mice. Transplantation of the FV-treated cells resulted in the successful reconstitution of functionally active T and B cells. These data suggest that FV vectors can be effective and may be safer than conventional RV vectors for gene therapy for SCID-X1.
Richardson Jason H
Full Text Available Abstract Background Several observations support the hypothesis that vector-driven selection plays an important role in shaping dengue virus (DENV genetic diversity. Clustering of DENV genetic diversity at a particular location may reflect underlying genetic structure of vector populations, which combined with specific vector genotype × virus genotype (G × G interactions may promote adaptation of viral lineages to local mosquito vector genotypes. Although spatial structure of vector polymorphism at neutral genetic loci is well-documented, existence of G × G interactions between mosquito and virus genotypes has not been formally demonstrated in natural populations. Here we measure G × G interactions in a system representative of a natural situation in Thailand by challenging three isofemale families from field-derived Aedes aegypti with three contemporaneous low-passage isolates of DENV-1. Results Among indices of vector competence examined, the proportion of mosquitoes with a midgut infection, viral RNA concentration in the body, and quantity of virus disseminated to the head/legs (but not the proportion of infected mosquitoes with a disseminated infection strongly depended on the specific combinations of isofemale families and viral isolates, demonstrating significant G × G interactions. Conclusion Evidence for genetic specificity of interactions in our simple experimental design indicates that vector competence of Ae. aegypti for DENV is likely governed to a large extent by G × G interactions in genetically diverse, natural populations. This result challenges the general relevance of conclusions from laboratory systems that consist of a single combination of mosquito and DENV genotypes. Combined with earlier evidence for fine-scale genetic structure of natural Ae. aegypti populations, our finding indicates that the necessary conditions for local DENV adaptation to mosquito vectors are met.
Liu, Si; Xie, Jiatao; Cheng, Jiasen; Li, Bo; Chen, Tao; Fu, Yanping; Li, Guoqing; Wang, Manqun; Jin, Huanan; Wan, Hu; Jiang, Daohong
Mycoviruses are usually transmitted horizontally via hyphal anastomosis and vertically via sexual/asexual spores. Previously, we reported that a gemycircularvirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), could infect its fungal host extracellularly. Here, we discovered that SsHADV-1 could infect a mycophagous insect, Lycoriella ingenua, and use it as a transmission vector. Virus acquired by larvae feeding on colonies of a virus-infected strain of S. sclerotiorum was replicated and retained in larvae, pupae, adults, and eggs. Virus could be transmitted to insect offspring when larvae were injected with virus particles and allowed to feed on a nonhost fungus. Virus replication in insect cells was further confirmed by inoculating Spodoptera frugiperda cells with virus particles and analyzing with RT-PCR, Northern blot, immunofluorescence, and flow cytometry assays. Larvae could transmit virus once they acquired virus by feeding on virus-infected fungal colony. Offspring larvae hatched from viruliferous eggs were virus carriers and could also successfully transmit virus. Virus transmission between insect and fungus also occurred on rapeseed plants. Virus-infected isolates produced less repellent volatile substances to attract adults of L. ingenua. Furthermore, L. ingenua was easily observed on Sclerotinia lesions in rapeseed fields, and viruliferous adults were captured from fields either sprayed with a virus-infected fungal strain or nonsprayed. Our findings may facilitate the exploration of mycoviruses for control of fungal diseases and enhance our understanding of the ecology of SsHADV-1 and other newly emerging SsHADV-1–like viruses, which were recently found to be widespread in various niches including human HIV-infected blood, human and animal feces, insects, plants, and even sewage. PMID:27791095
Kaneda, Yasufumi; Nakajima, Toshihiro; Nishikawa, Tomoyuki; Yamamoto, Seiji; Ikegami, Hiroyuki; Suzuki, Naho; Nakamura, Hitomi; Morishita, Ryuichi; Kotani, Hitoshi
We have developed a simple method for converting the lipid envelope of an inactivated virus to a gene transfer vector. Hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector was constructed by incorporating plasmid DNA into inactivated HVJ particles. This HVJ envelope vector introduced plasmid DNA efficiently and rapidly into various cell lines, including cancer cells and several types of primary cell culture. Efficiency of gene transfer was greatly enhanced by protamine sulfate and centrifugation. Fluorescein isothiocyanate-labeled oligodeoxynucleotides (FITC-ODN) were also delivered to cells at > 95% efficiency. When HVJ envelope vector was injected into organs directly, reporter gene expression was observed in organs including liver, brain, skin, uterus, tumor masses, lung, and eye. When HVJ envelope vector containing luciferase gene was injected into mouse tail vein, luciferase gene expression was detected primarily in spleen. FITC-ODN were also delivered to spleen cells by intravenous injection of HVJ envelope. These results suggest that HVJ envelope vector will be useful for both ex vivo and in vivo gene therapy experiments.
Chad E Mire
Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.
Ahmad, Tauqeer; AbouHaidar, Mounir; Hefferon, Kathleen L
Improved knowledge of the molecular biology of viruses, including recent gains in virus sequence data analysis, has greatly contributed to recent innovations in medical diagnostics, therapeutics, drug development and other related areas. Virus sequences have been used for the development of vaccines and antiviral agents to block the spread of viral infections, as well as to target and battle chronic diseases such as cancer. Virus sequences are now routinely employed in a wide array of RNA silencing technologies. Viruses can also be engineered into expression vectors which in turn can be used as protein production platforms as well as delivery vehicles for gene therapies. This review article outlines a number of patents that have been recently issued with respect to virus sequence data and describes some of their biotechnological applications.
Lindbo John A
Full Text Available Abstract Background Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use. Results We have constructed a Cauliflower mosaic virus (CaMV 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI. The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date. Conclusion These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.
Díaz Desani, Beatriz M.; Biurrun, R. (Ricardo); Moreno, Aránzazu; Nebreda, Miguel; Fereres, Alberto
Ultraviolet (UV)-absorbing plastic films are being used as a photoselective barrier to control insect vectors and associated virus diseases in different horticultural crops. A 2-year experiment was carried out in northeastern Spain (Navarra) to evaluate the impact of a UV-blocking film (AD-IR AV) on the population density of insect pests and the spread of insect-transmitted virus diseases associated with head lettuce [Lactuca sativa (L.)]. Results showed that the UV-absorbing plastic film did...
Pei-Sze Jeslyn Wong
Full Text Available Zika virus (ZIKV is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV.To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80-85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi. Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose50 assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious.The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.
Haberman, Rebecca; Criswell, Hugh; Snowdy, Stephen; Ming, Zhen; Breese, George; Samulski, R; McCown, Thomas
The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 microl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracyclineoff promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy.
Tabin, C J; Hoffmann, J W; Goff, S P; Weinberg, R A
We investigated the feasibility of using retroviruses as vectors for transferring DNA sequences into animal cells. The thymidine kinase (tk) gene of herpes simplex virus was chosen as a convenient model. The internal BamHI fragments of a DNA clone of Moloney leukemia virus (MLV) were replaced with a purified BamHI DNA segment containing the tk gene. Chimeric genomes were created carrying the tk insert in both orientations relative to the MLV sequence. Each was transfected into TK- cells along with MLV helper virus, and TK+ colonies were obtained by selection in the presence of hypoxanthine, aminopterin, and thymidine (HAT). Virus collected from TK+-transformed, MLV producer cells passed the TK+ phenotype to TK- cells. Nonproducer cells were isolated, and TK+ transducing virus was subsequently rescued from them. The chimeric virus showed single-hit kinetics in infections. Virion and cellular RNA and cellular DNA from infected cells were all shown to contain sequences which hybridized to both MLV- and tk-specific probes. The sizes of these sequences were consistent with those predicted for the chimeric virus. In all respects studied, the chimeric MLV-tk virus behaved like known replication-defective retroviruses. These experiments suggest great general applicability of retroviruses as eucaryotic vectors. Images PMID:6180306
Zhang, Jiachun; Guo, Xianfeng; Zhang, Min; Wu, Feifan; Peng, Yongzheng
To construct a universal, highly attenuated orf virus expression vector for exogenous genes using green fluorescent protein (GFP) as the reporter gene. The flanking regions of the ORFV132 of orf virus DNA were amplified by PCR to construct the shuttle plasmid pSPV-132LF-EGFP-132RF. The shuttle plasmid was transfected into OFTu cells and GFP was incorporated into orf virus IA82Delta 121 by homologous recombination. The recombinant IA82Delta121-V was selected by green fluorescent signal. The deletion gene was identified by PCR and sequencing. The effects of ORFV132 knockout were evaluated by virus titration and by observing the proliferation of the infected vascular endothelial cells in vitro. The recombinant orf virus IA82Delta121-V was obtained successfully and quickly, and the deletion of ORFV132 did not affect the replication of the virus in vitro but reduced its virulence. Green fluorescent protein is a selectable marker for rapid, convenient and stable selection of the recombinant viruses. Highly attenuated recombinant orf virus IA82Delta121-V can serve as a new expression vector for exogenous genes.
WU, Ping; Phillips, M. Ian; Bui, John; Terwilliger, Ernest F.
The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration...
Turner, Joanne; Bowers, Roger G.; Baylis, Matthew
Bluetongue is a notifiable disease of ruminants which, in 2007, occurred for the first time in England. We present the first model for bluetongue that explicitly incorporates farm to farm movements of the two main hosts, as well as vector dispersal. The model also includes a seasonal vector to host ratio and dynamic restriction zones that evolve as infection is detected. Batch movements of sheep were included by modelling degree of mixing at markets. We investigate the transmission of bluetongue virus between farms in eastern England (the focus of the outbreak). Results indicate that most parameters affecting outbreak size relate to vectors and that the infection generally cannot be maintained without between-herd vector transmission. Movement restrictions are effective at reducing outbreak size, and a targeted approach would be as effective as a total movement ban. The model framework is flexible and can be adapted to other vector-borne diseases of livestock. PMID:22432051
Schultz Ronald D
Full Text Available Abstract Background Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2 VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation. Results A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli can interrupt the downstream work.
Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...
Guayasamin, Ryann C; Reynolds, Tracy D; Wei, Xin; Fujiwara, Mai; Robek, Michael D
Vesicular stomatitis virus (VSV) has been extensively studied as a vaccine vector and oncolytic agent. Nevertheless, safety concerns have limited its widespread use in humans. The type III lambda interferon (IFN-λ) family of cytokines shares common signaling pathways with the IFN-α/β family and thus evokes similar antiviral activities. However, IFN-λ signals through a distinct receptor complex that is expressed in a cell type-specific manner, which restricts its activity to epithelial barriers, particularly those corresponding to the respiratory and gastrointestinal tracts. In this study, we determined how IFN-λ expression from recombinant VSV would influence vector replication, spread, and immunogenicity. We demonstrate that IFN-λ expression severely attenuates VSV in cell culture. In vivo, IFN-λ limits VSV replication in the mouse lung after intranasal administration and reduces virus spread to other organs. Despite this attenuation, however, the vector retains its capacity to induce protective CD8 T cell and antibody responses after a single immunization. These findings demonstrate a novel method of viral vector attenuation that could be used in both vaccine and oncolytic virus applications. Viruses such as VSV that are used as vaccine vectors can induce protective T cell and antibody responses after a single dose. Additionally, IFN-λ is a potent antiviral agent that has certain advantages for clinical use compared to IFN-α/β, such as fewer patient side effects. Here, we demonstrate that IFN-λ attenuates VSV replication and spread following intranasal virus delivery but does not reduce the ability of VSV to induce potent protective immune responses. These findings demonstrate that the type III IFN family may have widespread applicability for improving the safety and efficacy of viral vaccine and oncolytic vectors. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Full Text Available Zika virus (ZIKV has now become a global public health concern. The vectors for ZIKV are Aedes aegypti and Aedes albopictus. Both these mosquitoes are predominant in Southeast Asia and are also responsible for the spread of other arboviral diseases like dengue virus (DENV and chikungunya virus (CHIKV. The incidence of dengue has been increasing over the years and this is of concern to public health workers. Simple laboratory tools for the detection of ZIKV is also lacking. In the absence of drugs and vaccine for these arboviral diseases, vector control is the main option for surveillance and control. Aedes larval surveys have been the hallmark of dengue control along with larviciding and fogging when cases are reported. However, we need new paradigms and options for control of these vectors. The current situation in Southeast Asia clearly proves that effective strategies for vector control need to be proactive and not reactive. This will be the way forward to control epidemics of these diseases inclusive of ZIKV until a vaccine becomes available.
VSV (Simon, Cardomone et al. 1990), borna disease virus (Formella, Jehle et al. 2000), and Sinbis virus (Karpf, Lenches et al. 1997). The...C., et al. (2000). "Sequence variability of Borna disease virus : resistance to superinfection may contribute to high genome stability in...Marburg virus disease ". S Afr Med J 66(2):50-4 Roberts, A., L. Buonocore, et al. (1999). "Attenuated vesicular stomatitis viruses as vaccine vectors." J
Loot, AE; Henning, RH; Deelman, LE; Tio, RA; Schoen, P; Wilschut, JC; van Gijst, WH; Roks, AJM
Gene therapy is emerging as a realistic addition to the therapeutic arsenal in heart failure, but the search for suitable vectors for cardiac transfection is still ongoing. In this study, we explore the applicability of recombinant Semliki Forest virus (SFV) in heart failure. SFV was intracoronarily
Heidi E Brown; Alex Young; Joceline Lega; Theodore G Andreadis; Jessica Schurich; Andrew Comrie
... to infection, which varies among populations, and pathogen and vector development rates, which are influenced by weather (Hartvigsen et al. 1998; Paz and Semenza 2013). West Nile virus disease (WNV) is now the most common domestic arthropodborne viral disease (Reimann et al. 2008). Presumably introduced from the Middle East, the first WNV case in the...
Seppen, Jurgen; Bakker, Conny; de Jong, Berry; Kunne, Cindy; van den Oever, Karin; Vandenberghe, Kristin; de Waart, Rudi; Twisk, Jaap; Bosma, Piter
Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have
Apparailly, F.; Khoury, M.; Vervoordeldonk, M. J. B.; Adriaansen, J.; Gicquel, E.; Perez, N.; Riviere, C.; Louis-Plence, P.; Noel, D.; Danos, O.; Douar, A.-M.; Tak, P. P.; Jorgensen, C.
The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis ( RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues.
Potato virus Y (PVY) strains are transmitted by different aphid species in a non-persistent, non-circulative manner. Green peach aphid (GPA, Myzus persicae Sulzer; Aphididae, Macrosiphini) is the most efficient vector in laboratory studies, but potato aphid (PA, Macrosiphum euphorbiae Thomas; Aphidi...
D. Nanda (Dharminderkoemar); R. Vogels; M. Havenga; C.J.J. Avezaat (Cees); A. Bout; P.S. Smitt
textabstractWe evaluated the interaction between oncolytic, replication-competent adenoviral vectors and the herpes simplex virus-1 thymidine kinase (HSV1-tk) gene/ganciclovir (GCV) suicide system for the treatment of malignant gliomas. We constructed a panel of
Defective interfering particles (DIPs) have been found for many important viral pathogens and it is believed that most viruses generate DIPs. This article reviews the current knowledge of the generation and amplification of DIPs, which possess deletions in the viral genome but retain the ability to replicate in the presence of a complete helper virus. In addition, mechanisms are discussed by which DIPs interfere with the replication of their helper virus leading to the production of mainly progeny DIPs by coinfected cells. Even though DIPs cannot replicate on their own, they are biologically active and it is well known that they have a huge impact on virus replication, evolution, and pathogenesis. Moreover, defective genomes are potent inducers of the innate immune response. Yet, little attention has been paid to DIPs in recent years and their impact on biotechnological products such as vaccines and viral vectors remains elusive in most cases. With a focus on influenza virus, this review demonstrates that DIPs are important for basic research on viruses and for the production of viral vaccines and vectors. Reducing the generation and/or amplification of DIPs ensures reproducible results as well as high yields and consistent product quality in virus production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Lenzi, Andrea; Mirone, Vincenzo; Gentile, Vincenzo; Bartoletti, Riccardo; Ficarra, Vincenzo; Foresta, Carlo; Mariani, Luciano; Mazzoli, Sandra; Parisi, Saverio G; Perino, Antonio; Picardo, Mauro; Zotti, Carla Maria
Human Papillomavirus (HPV) is a very resistant, ubiquitous virus that can survive in the environment without a host. The decision to analyse HPV-related diseases in males was due to the broad dissemination of the virus, and, above all, by the need to stress the importance of primary and secondary prevention measures (currently available for women exclusively). The objective of the Consensus Conference was to make evidence-based recommendations that were designed to facilitate the adoption of a standard approach in clinical practice in Italy. The Sponsoring Panel put a series of questions to the members of the Scientific Committee who prepared a summary of the currently available information, relevant for each question, after the review and grading of the existing scientific literature. The summaries were presented to a Jury, also called multidisciplinary Consensus Panel, who drafted a series of recommendations. The prevalence of HPV in males ranges between 1.3-72.9%;. The prevalence curve in males is much higher than that in females and does not tend to decline with age. Women appear to have a higher probability of acquiring HPV genotypes associated with a high oncogenic risk, whereas in males the probability of acquiring low- or high-risk genotypes is similar. The HPV-related diseases that affect males are anogenital warts and cancers of the penis, anus and oropharynx. The quadrivalent vaccine against HPV has proved to be effective in preventing external genital lesions in males aged 16-26 years in 90.4%; (95%; CI: 69.2-98.1) of cases. It has also proved to be effective in preventing precancerous anal lesions in 77.5%; (95%; CI: 39.6-93.3) of cases in a per-protocol analysis and in 91.7%; (95%; CI: 44.6-99.8) of cases in a post-hoc analysis. Early ecological studies demonstrate reduction of genital warts in vaccinated females and some herd immunity in males when vaccine coverage is high, although males who have sex with males gained no benefit at all. Males with
McElroy, A K; Smith, J M; Hooper, J W; Schmaljohn, C S
Sin Nombre virus (SNV) and Andes virus (ANDV), members of the genus Hantavirus, in the family Bunyaviridae, are causative agents of hantavirus pulmonary syndrome (HPS) in North and South America, respectively. Although ANDV causes a lethal HPS-like disease in hamsters, SNV, and all other HPS-associated hantaviruses that have been tested, cause asymptomatic infections of laboratory animals, including hamsters. In an effort to understand the pathogenicity of ANDV in the hamster model, we generated ANDV/SNV reassortant viruses. Plaque isolation of viruses from cell cultures infected with both parental viruses yielded only one type of stable reassortant virus: large (L) and small (S) segments of SNV and M segment of ANDV. This virus, designated SAS reassortant virus, had in vitro growth and plaque morphology characteristics similar to those of ANDV. When injected into hamsters, the SAS reassortant virus was highly infectious and elicited high-titer, ANDV-specific neutralizing antibodies; however, the virus did not cause HPS and was not lethal. These data indicate that the ANDV M genome segment is not sufficient to confer the lethal HPS phenotype associated with ANDV.
Boeriis, Morten; van Leeuwen, Theo
This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...
Mascia, Tiziana; Nigro, Franco; Abdallah, Alì; Ferrara, Massimo; De Stradis, Angelo; Faedda, Roberto; Palukaitis, Peter; Gallitelli, Donato
RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.
Full Text Available The relationships between plant viruses, their herbivore vectors and host plants can be beneficial, neutral, or antagonistic, depending on the species involved. This variation in relationships may affect the process of biological invasion and the displacement of indigenous species by invaders when the invasive and indigenous organisms occur with niche overlap but differ in the interactions. The notorious invasive B biotype of the whitefly complex Bemisia tabaci entered China in the late 1990s and is now the predominant or only biotype in many regions of the country. Tobacco curly shoot virus (TbCSV and Tomato yellow leaf curl China virus (TYLCCNV are two whitefly-transmitted begomoviruses that have become widespread recently in south China. We compared the performance of the invasive B and indigenous ZHJ1 whitefly biotypes on healthy, TbCSV-infected and TYLCCNV-infected tobacco plants. Compared to its performance on healthy plants, the invasive B biotype increased its fecundity and longevity by 12 and 6 fold when feeding on TbCSV-infected plants, and by 18 and 7 fold when feeding on TYLCCNV-infected plants. Population density of the B biotype on TbCSV- and TYLCCNV-infected plants reached 2 and 13 times that on healthy plants respectively in 56 days. In contrast, the indigenous ZHJ1 performed similarly on healthy and virus-infected plants. Virus-infection status of the whiteflies per se of both biotypes showed limited effects on performance of vectors on cotton, a nonhost plant of the viruses. The indirect mutualism between the B biotype whitefly and these viruses via their host plants, and the apparent lack of such mutualism for the indigenous whitefly, may contribute to the ability of the B whitefly biotype to invade, the displacement of indigenous whiteflies, and the disease pandemics of the viruses associated with this vector.
Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.
Darpel, Karin E.; Langner, Kathrin F. A.; Nimtz, Manfred; Anthony, Simon J.; Brownlie, Joe; Takamatsu, Haru-Hisa; Mellor, Philip S.; Mertens, Peter P. C.
Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, ‘VP2’, can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent / non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2–6 fold. Treatment of an ‘eastern’ strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a ‘western’ strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to
Karin E Darpel
Full Text Available Bluetongue virus (BTV and epizootic haemorrhagic disease virus (EHDV are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.. The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin, forming infectious subviral particles (ISVP which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis. We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector, cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species. Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species, can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to
El-Mohtar, Choaa; Dawson, William O
We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3' terminus. Yet, this virus has 10 3'-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3' terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3' region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees. © 2013 Elsevier Inc. All rights reserved.
Wang, Feng-Xue; Liu, Ying; Zhu, Hong-Wei; Liu, Xing; Yang, Yong; Sun, Na; Cheng, Shi-Peng; Wen, Yong-Jun
Bovine parainfluenza virus 3 (BPIV3) is one of the most important respiratory pathogens in cattle. One BPIV3, named NM09, was isolated from cattle suffering from severe respiratory diseases in 2009. BPIV3 is a potential recombinant vaccine vector. To investigate whether NM09 can infect pigs and determine BPIV3 defense in these animals, BPIV3 antibody-free pigs were inoculated intramuscularly with the BPIV3 NM09 strain in a continuous passage. Clinical signs were observed each day after inoculation. Viral nucleic acid was detected in nasal and anal secretions. Results showed that virus-inoculated pigs displayed few observable clinical signs related to respiratory diseases. The antibody was identified, but the virus could not be detected in the second continuous passage in pigs. Thus, BPIV3 is a potential vaccine vector for genetic engineering.
Liu, Yarong; Siriwon, Natnaree; Rohrs, Jennifer A; Wang, Pin
Adeno-associated virus (AAV) vectors are promising human gene delivery vehicles due to their ability to establish long-term gene expression in a wide variety of target tissues; however, the broad native viral tropism raises concerns over the feasibility and safety of their systemic administration. To overcome this issue, much effort has been made to redirect AAVs toward specific tissues. This review presents several design strategies that have been applied to generate AAVs that target specific tissues and cells while inhibiting the transduction of non-target tissues. Multiple methods of vector capsid engineering have shown promise in vitro, including indirect targeting by adaptor systems and direct targeting by the insertion of antibodies or receptor-specific small peptide motifs. Other strategies, including creating mosaic or chimeric capsids and directed evolution, have also been used to successfully retarget AAV vectors. This research will further expand the clinical applications of AAV vectors by enhancing the control over tissue-specific gene delivery.
Wu, P; Phillips, M I; Bui, J; Terwilliger, E F
The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.
Zhang, Jianfeng; Jex, Edward; Feng, Tsungwei; Sivko, Gloria S; Baillie, Leslie W; Goldman, Stanley; Van Kampen, Kent R; Tang, De-chu C
Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine.
Full Text Available Viral vectors that can infect neurons transsynaptically and can strongly express foreign genes are useful for investigating the organization of neural circuits. We previously developed a propagation-competent rabies virus (RV vector based on a highly attenuated HEP-Flury strain (rHEP5.0-CVSG, which selectively infects neurons and propagates between synaptically connected neurons in a retrograde direction. Its relatively low level of transgene expression, however, makes immunostaining necessary to visualize the morphological features of infected neurons. To increase the transgene expression level of this RV vector, in this study we focused on two viral proteins: the large protein (L and matrix protein (M. We first attempted to enhance the expression of L, which is a viral RNA polymerase, by deleting the extra transcription unit and shortening the intergenic region between the G and L genes. This viral vector (rHEP5.0-GctL showed increased transgene expression level with efficient transsynaptic transport. We next constructed an RV vector with a rearranged gene order (rHEP5.0-GML with the aim to suppress the expression of M, which plays a regulatory role in virus RNA synthesis. Although this vector showed high transgene expression level, the efficiency of transsynaptic transport was low. To further evaluate the usability of rHEP5.0-GctL as a transsynaptic tracer, we inserted a fluorescent timer as a transgene, which changes the color of its fluorescence from blue to red over time. This viral vector enabled us the differentiation of primary infected neurons from secondary infected neurons in terms of the fluorescence wavelength. We expect this propagation-competent RV vector to be useful for elucidating the complex organization of the central nervous system.
Goodman, Michael Aaron; Arumugam, Paritha; Pillis, Devin Marie; Loberg, Anastacia; Nasimuzzaman, Mohammed; Lynn, Danielle; van der Loo, Johannes Christiaan Maria; Dexheimer, Phillip Joseph; Keddache, Mehdi; Bauer, Thomas Roy; Hickstein, Dennis Durand; Russell, David William; Malik, Punam
Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene
Michelle V Evans; Dallas, Tad; Han, Barbara A; Murdock, Courtney C.; John M. Drake
eLife digest Mosquitoes carry several diseases that pose an emerging threat to society. Outbreaks of these diseases are often sudden and can spread to previously unaffected areas. For example, the Zika virus was discovered in 1947, but only received international attention when it spread to the Americas in 2014, where it caused over 100,000 cases in Brazil alone. While we now recognize the threat Zika can pose for public health, our knowledge about the ecology of the disease remains poor. Nin...
Azar, Sasha R; Roundy, Christopher M; Rossi, Shannan L; Huang, Jing H; Leal, Grace; Yun, Ruimei; Fernandez-Salas, Ildefonso; Vitek, Christopher J; Paploski, Igor A D; Stark, Pamela M; Vela, Jeremy; Debboun, Mustapha; Reyna, Martin; Kitron, Uriel; Ribeiro, Guilherme S; Hanley, Kathryn A; Vasilakis, Nikos; Weaver, Scott C
To evaluate the potential role of Aedes albopictus (Skuse) as a vector of Zika virus (ZIKV), colonized mosquitoes of low generation number (≤ F5) from Brazil, Houston, and the Rio Grande Valley of Texas engorged on viremic mice infected with ZIKV strains originating from Senegal, Cambodia, Mexico, Brazil, or Puerto Rico. Vector competence was established by monitoring infection, dissemination, and transmission potential after 3, 7, and 14 days of extrinsic incubation. Positive saliva samples were assayed for infectious titer. Although all three mosquito populations were susceptible to all ZIKV strains, rates of infection, dissemination, and transmission differed among mosquito and virus strains. Aedes albopictus from Salvador, Brazil, were the least efficient vectors, demonstrating susceptibility to infection to two American strains of ZIKV but failing to shed virus in saliva. Mosquitoes from the Rio Grande Valley were the most efficient vectors and were capable of shedding all three tested ZIKV strains into saliva after 14 days of extrinsic incubation. In particular, ZIKV strain DakAR 41525 (Senegal 1954) was significantly more efficient at dissemination and saliva deposition than the others tested in Rio Grande mosquitoes. Overall, our data indicate that, while Ae. albopictus is capable of transmitting ZIKV, its competence is potentially dependent on geographic origin of both the mosquito population and the viral strain.
Rausalu, Kai; Iofik, Anna; Ulper, Liane; Karo-Astover, Liis; Lulla, Valeria; Merits, Andres
Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important. We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture. The properties of replication-competent vectors of alphaviruses depend on the details of their
Alfred O. Ochieng
Full Text Available Background: Rift Valley fever (RVF is a vector-borne zoonotic disease that has an impact on human health and animal productivity. Here, we explore the use of vector presence modelling to predict the distribution of RVF vector species under climate change scenario to demonstrate the potential for geographic spread of Rift Valley fever virus (RVFV. Objectives: To evaluate the effect of climate change on RVF vector distribution in Baringo County, Kenya, with an aim of developing a risk map for spatial prediction of RVF outbreaks. Methodology: The study used data on vector presence and ecological niche modelling (MaxEnt algorithm to predict the effect of climatic change on habitat suitability and the spatial distribution of RVF vectors in Baringo County. Data on species occurrence were obtained from longitudinal sampling of adult mosquitoes and larvae in the study area. We used present (2000 and future (2050 Bioclim climate databases to model the vector distribution. Results: Model results predicted potential suitable areas with high success rates for Culex quinquefasciatus, Culex univitattus, Mansonia africana, and Mansonia uniformis. Under the present climatic conditions, the lowlands were found to be highly suitable for all the species. Future climatic conditions indicate an increase in the spatial distribution of Cx. quinquefasciatus and M. africana. Model performance was statistically significant. Conclusion: Soil types, precipitation in the driest quarter, precipitation seasonality, and isothermality showed the highest predictive potential for the four species.
Sakkas, Hercules; Economou, Vangelis; Papadopoulou, Chrissanthy
Zika virus infection is an emerging mosquito-borne disease, first identified in Uganda in 1947. It is caused by the Zika arbovirus, and transmitted by the bites of infected mosquitoes of the genus Aedes. For almost half a century, the Zika virus was reported as the causative agent of sporadic human infections. In 2007, the Zika virus emerged outside Asia and Africa causing an epidemic on the Island of Yap in Micronesia. The manifestation of the newly acquired human infection varies from asymptomatic to self-limiting acute febrile illness with symptoms and clinical features similar to those caused by the Dengue virus ('Dengue-like syndrome'). The real-time PCR and serological methods have been successfully applied for the diagnosis of the disease. The treatment is symptomatic, since there is no specific antiviral treatment or a vaccine. During the recent outbreaks in French Polynesia and Brazil, incidents of Guillain-Barrι syndrome and microcephaly were associated with Zika virus infection, giving rise to fears of further global spread of the virus. Prevention and vector control strategies have to be urgently implemented by national health authorities in order to contain future outbreaks in vulnerable populations. This review summarizes the existing information on Zika virus characteristics, pathogenesis and epidemiology, the available methods for the diagnosis of Zika virus infection and recent approaches for prevention and control.
Full Text Available In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas.
Weger-Lucarelli, James; Rückert, Claudia; Chotiwan, Nunya; Nguyen, Chilinh; Garcia Luna, Selene M; Fauver, Joseph R; Foy, Brian D; Perera, Rushika; Black, William C; Kading, Rebekah C; Ebel, Gregory D
In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas.
Full Text Available Over 60,500 dengue cases were reported in the state of Espírito Santo (ES, Brazil, between 1995 and 1998. The study's purpose was to identify whether Aedes albopictus was transmitting the dengue virus during an epidemic in the locality of Vila Bethânia (Viana County,Vitória, ES. From April 3 to 9, 1998, blood and serum samples were collected daily for virus isolation and serological testing. Four autochthonous cases were confirmed through DEN 1 virus isolation and two autochthonous cases through MAC ELISA testing. Of 37 Ae. aegypti and 200 Ae. albopictus adult mosquitoes collected and inoculated, DEN1 virus was isolated only from a pool of two Ae. aegypti female mosquitoes. The study results suggest that Ae. albopictus still cannot be considered an inter-human vector in dengue epidemics in Brazil.
Wollmann, Guido; Paglino, Justin C.; Maloney, Patrick R; Ahmadi, Sebastian A; van den Pol, Anthony N
Vesicular stomatitis virus (VSV) shows promise as vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-D-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain. PMID:25462341
Wollmann, Guido; Paglino, Justin C; Maloney, Patrick R; Ahmadi, Sebastian A; van den Pol, Anthony N
Vesicular stomatitis virus (VSV) shows promise as a vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-d-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of IFN, ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain. Copyright © 2014 Elsevier Inc. All rights reserved.
Full Text Available Barley stripe mosaic virus (BSMV is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS, magnesium chelatase subunit H (ChlH, and plastid transketolase (TK gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5 also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.
Linz, Lucas B.; LIU, Sijun; Chougule, Nanasaheb P.; Bonning, Bryony C.
Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and ...
Full Text Available Dengue virus genotypes of Southeast Asian origin have been associated with higher virulence and transmission compared to other genotypes of serotype 2 (DEN-2. We tested the hypothesis that genetic differences in dengue viruses may result in differential binding to the midgut of the primary vector, Aedes aegypti, resulting in increased transmission or vectorial capacity.Two strains of each of the four DEN-2 genotypes (Southeast Asian, American, Indian, and West African were tested to determine their binding affinity for mosquito midguts from two distinct populations (Tapachula, Chiapas, Mexico and McAllen, Texas, USA. Our previous studies demonstrated that Southeast Asian viruses disseminated up to 65-fold more rapidly in Ae. aegypti from Texas and were therefore more likely to be transmitted to humans. Results shown here demonstrate that viruses from all four genotypes bind to midguts at the same rate, in a titer-dependent manner. In addition, we show population differences when comparing binding affinity for DEN-2 between the Tapachula and McAllen mosquito colonies.If midgut binding potential is the same for all DEN-2 viruses, then viral replication differences in these tissues and throughout the mosquito can thus probably explain the significant differences in dissemination and vector competence. These conclusions differ from the established paradigms to explain mosquito barriers to infection, dissemination, and transmission.
Cox, Jonathan; Brown, Heidi E; Rico-Hesse, Rebeca
Dengue virus genotypes of Southeast Asian origin have been associated with higher virulence and transmission compared to other genotypes of serotype 2 (DEN-2). We tested the hypothesis that genetic differences in dengue viruses may result in differential binding to the midgut of the primary vector, Aedes aegypti, resulting in increased transmission or vectorial capacity. Two strains of each of the four DEN-2 genotypes (Southeast Asian, American, Indian, and West African) were tested to determine their binding affinity for mosquito midguts from two distinct populations (Tapachula, Chiapas, Mexico and McAllen, Texas, USA). Our previous studies demonstrated that Southeast Asian viruses disseminated up to 65-fold more rapidly in Ae. aegypti from Texas and were therefore more likely to be transmitted to humans. Results shown here demonstrate that viruses from all four genotypes bind to midguts at the same rate, in a titer-dependent manner. In addition, we show population differences when comparing binding affinity for DEN-2 between the Tapachula and McAllen mosquito colonies. If midgut binding potential is the same for all DEN-2 viruses, then viral replication differences in these tissues and throughout the mosquito can thus probably explain the significant differences in dissemination and vector competence. These conclusions differ from the established paradigms to explain mosquito barriers to infection, dissemination, and transmission.
Gentle, J. N., Jr.; Kahn, A.; Pierce, S. A.; Wang, S.; Wade, C.; Moran, S.
With the continued spread of the zika virus in the United States in both Florida and Virginia, increased public awareness, prevention and targeted prediction is necessary to effectively mitigate further infection and propagation of the virus throughout the human population. The goal of this project is to utilize publicly accessible data and HPC resources coupled with machine learning algorithms to identify potential threat vectors for the spread of the zika virus in Texas, the United States and globally by correlating available zika case data collected from incident reports in medical databases (e.g., CDC, Florida Department of Health) with known bodies of water in various earth science databases (e.g., USGS NAQWA Data, NASA ASTER Data, TWDB Data) and by using known mosquito population centers as a proxy for trends in population distribution (e.g., WHO, European CDC, Texas Data) while correlating historical trends in the spread of other mosquito borne diseases (e.g., chikungunya, malaria, dengue, yellow fever, west nile, etc.). The resulting analysis should refine the identification of the specific threat vectors for the spread of the virus which will correspondingly increase the effectiveness of the limited resources allocated towards combating the disease through better strategic implementation of defense measures. The minimal outcome of this research is a better understanding of the factors involved in the spread of the zika virus, with the greater potential to save additional lives through more effective resource utilization and public outreach.
Ruder Mark G
Full Text Available Abstract Background Culicoides sonorensis (Diptera: Ceratopogonidae is a vector of epizootic hemorrhagic disease virus (EHDV serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD and other wild ruminants. Although historically rare, reports of clinical EHDV infection in cattle have increased in some parts of the world over the past decade. In 2006, an EHDV-7 epizootic in cattle resulted in economic loss for the Israeli dairy industry. White-tailed deer are susceptible to EHDV-7 infection and disease; however, this serotype is exotic to the US and the susceptibility of C. sonorensis to this cattle-virulent EHDV is not known. The objective of the study was to determine if C. sonorensis is susceptible to EHDV-7 infection and is a competent vector. Methods To evaluate the susceptibility of C. sonorensis, midges were fed on EHDV-7 infected WTD, held at 22 ± 1°C, and processed individually for virus isolation and titration on 4–16 days post feeding (dpf. Midges with a virus titer of ≥102.7 median tissue culture infective doses (TCID50/midge were considered potentially competent. To determine if infected C. sonorensis were capable of transmitting EHDV-7 to a host, a susceptible WTD was then fed on by a group of 14–16 dpf midges. Results From 4–16 dpf, 45% (156/350 of midges that fed on WTD with high titer viremia (>107 TCID50/ml were virus isolation-positive, and starting from 10–16 dpf, 32% (35/109 of these virus isolation-positive midges were potentially competent (≥102.7 TCID50/midge. Midges that fed on infected deer transmitted the virus to a susceptible WTD at 14–16 dpf. The WTD developed viremia and severe clinical disease. Conclusion This study demonstrates that C. sonorensis is susceptible to EHDV-7 infection and can transmit the virus to susceptible WTD, thus, C. sonorensis should be considered a potential vector of EHDV-7. Together with previous work, this study demonstrates
Eutimio Gustavo Fernández Núñez
Full Text Available The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.
Schrag, Stephanie J.; Rota, Paul A.; Bellini, William J.
High mutation rates typical of RNA viruses often generate a unique viral population structure consisting of a large number of genetic microvariants. In the case of viral pathogens, this can result in rapid evolution of antiviral resistance or vaccine-escape mutants. We determined a direct estimate of the mutation rate of measles virus, the next likely target for global elimination following poliovirus. In a laboratory tissue culture system, we used the fluctuation test method of estimating mutation rate, which involves screening a large number of independent populations initiated by a small number of viruses each for the presence or absence of a particular single point mutation. The mutation we focused on, which can be screened for phenotypically, confers resistance to a monoclonal antibody (MAb 80-III-B2). The entire H gene of a subset of mutants was sequenced to verify that the resistance phenotype was associated with single point mutations. The epitope conferring MAb resistance was further characterized by Western blot analysis. Based on this approach, measles virus was estimated to have a mutation rate of 9 × 10−5 per base per replication and a genomic mutation rate of 1.43 per replication. The mutation rates we estimated for measles virus are comparable to recent in vitro estimates for both poliovirus and vesicular stomatitis virus. In the field, however, measles virus shows marked genetic stability. We briefly discuss the evolutionary implications of these results. PMID:9847306
The conference brought together researchers working on virus structure and virus assembly in diverse systems. Information was integrated from many viral systems, including plant bacterial and eukaryotic viruses, and many techniques such as biophysical approaches of x-ray diffraction, electron microscopy and spectroscopy, along with molecular biological and molecular genetic analysis.
Perabo, Luca; Büning, Hildegard; Kofler, David M; Ried, Martin U; Girod, Anne; Wendtner, Clemens M; Enssle, Jörg; Hallek, Michael
Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.
Haberman, Rebecca P; McCown, Thomas J
Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application. Copyright 2002 Elsevier Science (USA)
Kato, Ryuichi; Wolfe, Darren; Coyle, Christian H.; Wechuck, James B.; Tyagi, Pradeep; Tsukamoto, Taiji; Nelson, Joel B.; Glorioso, Joseph C.; Chancellor, Michael B.; Yoshimura, Naoki
Summary Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrphic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20μl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein (GFP) and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before sacrifice to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared to untreated or control vector treated groups. The HSV-NTN group had more FG-positive MPG neurons than control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for improvement of erectile dysfunction following cavernous nerve injury. PMID:18668142
Liu, Te-Hui; Oscherwitz, Jon; Schnepp, Bruce; Jacobs, Jana; Yu, Fen; Cease, Kemp B; Johnson, Philip R.
Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments enc...
Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H
Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.
Martins, Mathias; Joshi, Lok R; Rodrigues, Fernando S; Anziliero, Deniz; Frandoloso, Rafael; Kutish, Gerald F; Rock, Daniel L; Weiblen, Rudi; Flores, Eduardo F; Diel, Diego G
The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV∆024RABV-G or ORFV∆121RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV∆024RABV-G and ORFV∆121RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV∆121RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV∆024RABV-G-immunized animals, indicating a higher immunogenicity of ORFVΔ121-based vectors in these animal species. Copyright © 2017 Elsevier Inc. All rights reserved.
Sakai, Makoto; Fujimoto, Naohiro; Ishii, Katsunori; Nakamura, Hiroyuki; Kaneda, Yasufumi; Awazu, Kunio
Photodynamic therapy (PDT) is a photochemical modality approved for cancer treatment. PDT has demonstrated efficacy in early stage lung cancer and esophageal cancer. The accumulation of photosensitizers in cancer cells is necessary to enhance the therapeutic benefits of PDT; however, photosensitizers have low uptake efficiency. To overcome this limitation, a drug delivery system, such as the hemagglutinating virus of Japan envelope (HVJ-E) vector, is required. In this study, the combination of PDT and HVJ-E was investigated for enhancing the efficacy of PDT. The photosensitizers that were evaluated included 5-aminolaevulinic acid (5-ALA), protoporphyrin IX (PPIX), and HVJ-PPIX. The uptake of the photosensitizers as increased twenty-fold with the addition of HVJ-E. The cytotoxicity of conventional 5-ALA was enhanced by the addition of HVJ-E vector. In conclusion, HVJ-E vector improved the uptake of photosensitizers and the PDT effect.
Toi, Cheryl S; Webb, Cameron E; Haniotis, John; Clancy, John; Doggett, Stephen L
There are many gaps to be filled in our understanding of mosquito-borne viruses, their relationships with vectors and reservoir hosts, and the environmental drivers of seasonal activity. Stratford virus (STRV) belongs to the genus Flavivirus and has been isolated from mosquitoes and infected humans in Australia but little is known of its vector and reservoir host associations. A total of 43 isolates of STRV from mosquitoes collected in New South Wales between 1995 and 2013 was examined to determine the genetic diversity between virus isolates and their relationship with mosquito species. The virus was isolated from six mosquito species; Aedes aculeatus, Aedes alternans, Aedes notoscriptus, Aedes procax, Aedes vigilax, and Anopheles annulipes. While there were distinct differences in temporal and spatial activity of STRV, with peaks of activity in 2006, 2010 and 2013, a sequence homology of 95.9%-98.4% was found between isolates and the 1961 STRV prototype with 96.2%-100% identified among isolates. Temporal differences but no apparent nucleotide divergence by mosquito species or geographic location was evident. The result suggests the virus is geographically widespread in NSW (albeit only from coastal regions) and increased local STRV activity is likely to be driven by reservoir host factors and local environmental conditions influencing vector abundance. While STRV may not currently be associated with major outbreaks of human disease, with the potential for urbanisation and climate change to increase mosquito-borne disease risks, and the possibility of genomic changes which could produce pathogenic strains, understanding the drivers of STRV activity may assist the development of strategic response to public health risks posed by zoonotic flaviviruses in Australia.
Fujino, Kan; Yamamoto, Yusuke; Daito, Takuji; Makino, Akiko; Honda, Tomoyuki; Tomonaga, Keizo
Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses. © 2017 The Societies and John Wiley & Sons Australia, Ltd.
Zhang, Jianfeng; Tarbet, E Bart; Toro, Haroldo; Tang, De-chu C
The disease-fighting power of vaccines has been a public health bonanza credited with the worldwide reduction of mortality and morbidity. The goal to further amplify its power by boosting vaccine coverage requires the development of a new generation of rapid-response vaccines that can be mass produced at low costs and mass administered by nonmedical personnel. The new vaccines also have to be endowed with a higher safety margin than that of conventional vaccines. The nonreplicating adenovirus-vectored vaccine holds promise in boosting vaccine coverage because the vector can be rapidly manufactured in serum-free suspension cells in response to a surge in demand, and noninvasively administered by nasal spray into human subjects in compliance with evolutionary medicine. In contrast to parenteral injection, noninvasive mucosal vaccination minimizes systemic inflammation. Moreover, pre-existing adenovirus immunity does not interfere appreciably with the potency of an adenovirus-vectored nasal vaccine. Nasal administration of adenovirus vectors encoding pathogen antigens is not only fear-free and painless, but also confers rapid and sustained protection against mucosal pathogens as a drug-vaccine duo since adenovirus particles alone without transgene expression can induce an anti-influenza state in the airway. In addition to human vaccination, animals can also be mass immunized by this class of vectored vaccines.
Reeves, Will K; Miller, Myrna M
We investigated the susceptibility of Culicoides sonorensis to Cache Valley virus (CVV) (family Bunyaviridae, genus Orthobunyavirus) infection and the potential that it could be a vector or site of virus reassortment. CVV is native to the New World and causes disease in livestock. Infected blood meals were fed to both a competent vector, Anopheles quadrimaculatus, and Culicoides sonorensis. All Anopheles mosquitoes were infected as expected, but only 21 % of the C. sonorensis insects were susceptible to infection. These appeared to present a midgut barrier, because virus persisted but did not disseminate. This means Culicoides sonorensis is not likely to be a vector of CVV but could be involved in viral reassortment. Schmallenberg virus (SBV) (family Bunyaviridae, genus Orthobunyavirus) was recently discovered in Europe and probably is a novel virus resulting from a reassortment of two orthobunyaviruses, and an ongoing epizootic in cattle and small ruminants has caused significant economic damage.
Haller, Aurelia A.; Miller, Tessa; Mitiku, Misrach; Coelingh, Kathleen
Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39°C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37°C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses. PMID:11090161
Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S; Chen, Qiang
Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for the generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties owing to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that are effective, safe, low cost, and amenable to large-scale manufacturing. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
Asjes C J; Blom-Barnhoorn G L
In this study control of spread by insect vectors of non-persistent Lily symptomless virus and Lily mottle virus in lily, Tulip breaking virus in tulip, Iris mild mosaic virus, Narcissus latent virus...
da Silva, Suzianny Maria Bezerra Cabral; Lavander, Henrique David; de Santana Luna, Manuella Maria; de Melo Eloi da Silva, Ana Odete; Gálvez, Alfredo Olivera; Coimbra, Maria Raquel Moura
In 2004, the infectious myonecrosis virus (IMNV) was recognized as the main cause of Litopenaeusvannamei shrimp culture's drop in Brazil. In health animal control programs, in order to reduce virus prevalence in production units it is necessary to screen live feed used. Among live diets used in aquaculture, the brine shrimp Artemia sp. is essential in crustacean larviculture and maturation. The aim of the present study was to investigate the susceptibility of Artemiafranciscana to IMNV through an immersion challenge and virus-phytoplankton adhesion route and to elucidate its role as a vector for IMNV transmission to L.vannamei. A. franciscana adults were infected with IMNV through both routes, as demonstrated by PCR-positive reactions. However, infected A. franciscana showed no signs of infection. More than 40% of L. vannamei juveniles fed with IMNV-infected A. franciscana by virus-phytoplankton adhesion route were positive by real-time PCR, whereas only a 10% infection rate was found among shrimp fed with IMNV-infected brine shrimp using the immersion challenge. Significant differences were found in mean viral load between immersion and virus-phytoplankton adhesion shrimp treatments (p ⩽ 0.05). Moreover, the mean viral loads were 1.34 × 10(2) and 1.48 × 10(4) copies/μg(-1) of total RNA for virus-phytoplankton adhesion and IMNV-infected tissue treatments, respectively, and the difference was not significant (p ⩾ 0.05). The results indicated that A. franciscana act as a vector for IMNV transmission under the experimental conditions examined. Although no mass mortalities were detected in L. vannamei fed with IMNV-infected brine shrimp, these infected shrimp should not be disregarded as a source of IMNV in grow-out units. Copyright © 2015 Elsevier Inc. All rights reserved.
Full Text Available BACKGROUND: The recent unprecedented emergence of arboviruses transmitted by Culicoides biting midges in northern Europe has necessitated the development of techniques to differentiate competent vector species. At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR data in the form of Cq values used to infer the presence of viral RNA in samples. METHODOLOGY/PRINCIPAL FINDINGS: This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR. CONCLUSIONS/SIGNIFICANCE: When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system.
Seppen, Jurgen; Bakker, Conny; de Jong, Berry; Kunne, Cindy; van den Oever, Karin; Vandenberghe, Kristin; de Waart, Rudi; Twisk, Jaap; Bosma, Piter
Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.
THOMAS AND OTHERS ENHANCED SURVEILLANCE FOR DENGUE Improving Dengue Virus Capture Rates in Humans and Vectors in Kamphaeng Phet Province...of Medical Sciences, Bangkok, Thailand. Abstract. Dengue is of public health importance in tropical and sub-tropical regions. Dengue virus (DENV...with confirmed dengue (initiates) and associated cluster individuals (associates) with entomologic sampling. A total of 438 associates were enrolled
Shrestha, Anita; Champagne, Donald E; Culbreath, Albert K; Rotenberg, Dorith; Whitfield, Anna E; Srinivasan, Rajagopalbabu
Persistent propagative viruses maintain intricate interactions with their arthropod vectors. In this study, we investigated the transcriptome-level responses associated with a persistent propagative phytovirus infection in various life stages of its vector using an Illumina HiSeq sequencing platform. The pathosystem components included a Tospovirus, Tomato spotted wilt virus (TSWV), its insect vector, Frankliniella fusca (Hinds), and a plant host, Arachis hypogaea (L.). We assembled (de novo) reads from three developmental stage groups of virus-exposed and non-virus-exposed F. fusca into one transcriptome consisting of 72 366 contigs and identified 1161 differentially expressed (DE) contigs. The number of DE contigs was greatest in adults (female) (562) when compared with larvae (first and second instars) (395) and pupae (pre- and pupae) (204). Upregulated contigs in virus-exposed thrips had blastx annotations associated with intracellular transport and virus replication. Upregulated contigs were also assigned blastx annotations associated with immune responses, including apoptosis and phagocytosis. In virus-exposed larvae, Blast2GO analysis identified functional groups, such as multicellular development with downregulated contigs, while reproduction, embryo development and growth were identified with upregulated contigs in virus-exposed adults. This study provides insights into differences in transcriptome-level responses modulated by TSWV in various life stages of an important vector, F. fusca.
Japanese encephalitis (JE) is a vector-borne disease caused by the Japanese encephalitis virus (JEV) that affects humans in Eastern and Southeastern Asia. Although it could be prevented by a vaccine, JE has no treatment and the inadvertent introduction of the virus into JEV-free countries, such as t...
Campbell, P R; Cremer, J E; Roach, R L; Steele, V; Subramaniam, S; Sivasubramaniam, V; Monsour, C; Mullins, T; Persley, D M; Gambley, C F
The Bowen region of Northern Queensland is an important winter production area for tomatoes in Australia. There are three economically important viruses in the region that affect tomato, Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Potato leafroll virus (PLRV), which are vectored by whiteflies, thrips and aphids, respectively. An area wide management approach is required to lower the primary inoculum throughout the district. To this end, we undertook investigations into the virus incidence and alternative hosts for the virus and vectors in different cropping regions throughout the district, as well as local management options such as insecticide application and possible non-host cover crops for the wet-season break in production. The initial incidence of Potato leafroll virus was very high, most probably due to abnormal weather patterns for the district, and has ceased to be a problem. Tomato yellow leaf curl virus is a continual problem even at the beginning of the season, indicating large reservoir host(s) in the environment. Only four alternative hosts have been identified: Stachytarpheta jamaicensis (TSWV), Solanum americanum (PLRV and TYLCV) Trianthema portulacastrum (TYLCV), and Amaranthus viridis(TLYCV). Different insecticide and application options were trialled for protection against Tomato yellow leaf curl virus, with the best possible option yielding marketable fruit more than ninety percent of a resistant hybrid. A trial of yield vs time of infection of TYLCV found that whitefly exclusion for 6 weeks post-transplant yielded an average increase of nearly three kilograms of marketable fruit per plant. A number of pulse crops have been confirmed as non-hosts of tomato yellow leaf curl for use as cover crops in the wet-season break. Most of the production has moved to dual resistant TYLCV/TSWV hybrids, though an area wide management program still needs to be established to reduce the primary inoculum throughout the district
Caley, I J; Betts, M R; Irlbeck, D M; Davis, N L; Swanstrom, R; Frelinger, J A; Johnston, R E
A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. T...
Dáder, Beatriz; Legarrea, Saioa; Moreno, Aránzazu; Plaza, María; Carmo-Sousa, Michele; Amor, Fermín; Viñuela, Elisa; Fereres, Alberto
Long-lasting insecticide-treated nets (LLITNs) constitute a novel alternative that combines physical and chemical tactics to prevent insect access and the spread of insect-transmitted plant viruses in protected enclosures. This approach is based on a slow-release insecticide-treated net with large hole sizes that allow improved ventilation of greenhouses. The efficacy of a wide range of LLITNs was tested under laboratory conditions against Myzus persicae, Aphis gossypii and Bemisia tabaci. Two nets were selected for field tests under a high insect infestation pressure in the presence of plants infected with Cucumber mosaic virus and Cucurbit aphid-borne yellows virus. The efficacy of Aphidius colemani, a parasitoid commonly used for biological control of aphids, was studied in parallel field experiments. LLITNs produced high mortality of aphids, although their efficacy decreased over time with sun exposure. Certain nets excluded whiteflies under laboratory conditions; however, they failed in the field. Nets effectively blocked the invasion of aphids and reduced the incidence of viruses in the field. The parasitoid A. colemani was compatible with LLITNs. LLITNs of appropriate mesh size can become a very valuable tool in combination with biocontrol agents for additional protection against insect vectors of plant viruses under IPM programmes. © 2014 Society of Chemical Industry.
Tomczyk, Tomasz; Orzechowska, Beata
Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, is a promising candidate for potential use in construction of antiviral vaccines. In the natural environment VSV is a pathogen of wild ungulates and livestock. Some of the features that make VSV an excellent platform for the development of a range of viral therapeutics includes its immunogenicity and ability to grow to high titers in cell lines approved for vaccine use. Infection in humans is rare and usually asymptomatic, with mild flu-like symptoms. Moreover, due to affinity of VSV envelope glycoprotein to the LDL (low-density lipoprotein) receptor, VSV is effective at targeting a variety of tissues in vivo. A series of research results confirm the possibility of developing VSV-based vaccines against human papilloma viruses (HPV), human immunodeficiency virus (HIV), hepatitis B virus (HBV) and filoviruses (MARV, ZEBOV and SEBOV), as well as the potential use of a successfully developed vaccine against hepatitis C virus (HCV). VSV is neurotropic and infection can cause a viral encephalitis in experimental animals. Therefore, intensive studies are being undertaken to achieve satisfactory expression of the viral antigens while maintaining the safety of the constructed vectors.
Maria R. Mendoza
Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.
Mendoza, Maria R; Payne, Alexandria N; Castillo, Sean; Crocker, Megan; Shaw, Brian D; Scholthof, Herman B
Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies) it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV) referred to as TG, and Tobacco mosaic virus (TMV) annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.
Mélissanne de Wispelaere
Full Text Available Japanese encephalitis virus (JEV is the causative agent of Japanese encephalitis, the leading cause of viral encephalitis in Asia. JEV transmission cycle involves mosquitoes and vertebrate hosts. The detection of JEV RNA in a pool of Culex pipiens caught in 2010 in Italy raised the concern of a putative emergence of the virus in Europe. We aimed to study the vector competence of European mosquito populations, such as Cx. pipiens and Aedes albopictus for JEV genotypes 3 and 5.After oral feeding on an infectious blood meal, mosquitoes were dissected at various times post-virus exposure. We found that the peak for JEV infection and transmission was between 11 and 13 days post-virus exposure. We observed a faster dissemination of both JEV genotypes in Ae. albopictus mosquitoes, when compared with Cx. pipiens mosquitoes. We also dissected salivary glands and collected saliva from infected mosquitoes and showed that Ae. albopictus mosquitoes transmitted JEV earlier than Cx. pipiens. The virus collected from Ae. albopictus and Cx. pipiens saliva was competent at causing pathogenesis in a mouse model for JEV infection. Using this model, we found that mosquito saliva or salivary glands did not enhance the severity of the disease.In this study, we demonstrated that European populations of Ae. albopictus and Cx. pipiens were efficient vectors for JEV transmission. Susceptible vertebrate species that develop high viremia are an obligatory part of the JEV transmission cycle. This study highlights the need to investigate the susceptibility of potential JEV reservoir hosts in Europe, notably amongst swine populations and local water birds.
de Wispelaere, Mélissanne; Desprès, Philippe; Choumet, Valérie
Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis, the leading cause of viral encephalitis in Asia. JEV transmission cycle involves mosquitoes and vertebrate hosts. The detection of JEV RNA in a pool of Culex pipiens caught in 2010 in Italy raised the concern of a putative emergence of the virus in Europe. We aimed to study the vector competence of European mosquito populations, such as Cx. pipiens and Aedes albopictus for JEV genotypes 3 and 5. After oral feeding on an infectious blood meal, mosquitoes were dissected at various times post-virus exposure. We found that the peak for JEV infection and transmission was between 11 and 13 days post-virus exposure. We observed a faster dissemination of both JEV genotypes in Ae. albopictus mosquitoes, when compared with Cx. pipiens mosquitoes. We also dissected salivary glands and collected saliva from infected mosquitoes and showed that Ae. albopictus mosquitoes transmitted JEV earlier than Cx. pipiens. The virus collected from Ae. albopictus and Cx. pipiens saliva was competent at causing pathogenesis in a mouse model for JEV infection. Using this model, we found that mosquito saliva or salivary glands did not enhance the severity of the disease. In this study, we demonstrated that European populations of Ae. albopictus and Cx. pipiens were efficient vectors for JEV transmission. Susceptible vertebrate species that develop high viremia are an obligatory part of the JEV transmission cycle. This study highlights the need to investigate the susceptibility of potential JEV reservoir hosts in Europe, notably amongst swine populations and local water birds.
Full Text Available Japanese encephalitis virus (JEV is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP strategy based on bamboo mosaic virus (BaMV for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.
Chen, Tsung-Hsien; Hu, Chung-Chi; Liao, Jia-Teh; Lee, Yi-Ling; Huang, Ying-Wen; Lin, Na-Sheng; Lin, Yi-Ling; Hsu, Yau-Heiu
Japanese encephalitis virus (JEV) is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP) strategy based on bamboo mosaic virus (BaMV) for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII) at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.
Lai, Chao-Han; Wang, Kuan-Chieh; Kuo, Cheng-Hsiang; Lee, Fang-Tzu; Cheng, Tsung-Lin; Chang, Bi-Ing; Yang, Yu-Jen; Shi, Guey-Yueh; Wu, Hua-Lin
Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl2-induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl2-induced AAA model and angiotensin II-infused AAA model, respectively. In the CaCl2-induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (10(11) genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development. Copyright © 2017 Elsevier B.V. All rights reserved.
Full Text Available Mosquitoes (Diptera: Culicidae represent a key threat for millions of humans and animals worldwide, since they act as vectors for important parasites and pathogens, including malaria, filariasis and a wide number of arboviruses. The recent outbreaks of Zika virus infections occurring in South America, Central America, and the Caribbean, represent the most recent four arrivals of important arboviruses in the western hemisphere, over the last 20 years, namely dengue, West Nile virus, and chikungunya. Since there are no specific treatments for Zika virus and the other arboviruses mentioned above, it should be highlighted that the eco-friendly and effective control of mosquito vectors is of pivotal importance. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in presence of ultra-low quantities of green-synthesized nanoparticles, which magnify their predation efficiency. Furthermore, behaviour-based control tools relying on the employ of swarming behaviour manipulation (i.e. the “lure and kill” approach, pheromone traps, sound traps need further research attention. In particular, detailed basic information on the physical and chemical cues routing mosquito swarming and mating dynamics is urgently required.
Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi
First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.
Full Text Available The current method for creation of vaccinia virus (VACV vectors involves using a selection and purification marker, however inclusion of a gene without therapeutic value in the resulting vector is not desirable for clinical use. The Cre-LoxP system has been used to make marker-free Poxviruses, but the efficiency was very low. To obtain a marker-free VACV vector, we developed marker gene excision systems to modify the thymidine kinase (TK region and N1L regions using Cre-Loxp and Flp-FRET systems respectively. CRISPR-Cas9 system significantly resulted in a high efficiency (â¼90% in generation of marker gene-positive TK-mutant VACV vector. The marker gene (RFP could be excised from the recombinant virus using Cre recombinase. To make a marker-free VV vector with double gene deletions targeting the TK and N1L gene, we constructed a donor repair vector targeting the N1L gene, which can carry a therapeutic gene and the marker (RFP that could be excised from the recombinant virus using Flp recombinase. The marker-free system developed here can be used to efficiently construct VACV vectors armed with any therapeutic genes in the TK region or N1L region without marker genes. Our marker-free system platform has significant potential for development of new marker-free VACV vectors for clinical application.
Full Text Available Lentiviral vectors (LVs are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV glycoproteins, the hemagglutinin (H, responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV in presence of α-MV antibody-positive human plasma. At plasma dilution 1:160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1:80 an at least 4-times higher multiplicity of infection (MOI of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are
Kant, Ravi; Dasgupta, Indranil
Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v /F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.
Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C Kathy; Jin, Hong
Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1(st) or the 3(rd) position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3(rd) position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation.
Kurosaki, F; Uchibori, R; Mato, N; Sehara, Y; Saga, Y; Urabe, M; Mizukami, H; Sugiyama, Y; Kume, A
An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken β-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.
Direct measurements of the strength of particle interactions are critical for characterizing the stability and behavior of colloidal and nanoparticle suspensions. Current techniques are limited in their ability to measure pico-newton scale interaction forces on sub-micrometer particles due to signal detection limits, thermal noise, and throughput. We have recently developed a technique for making direct mechanical measurements of the force and work associated with the steric and electrostatic effects that stabilize colloidal nanoparticles. "Nanophotonic Force Microscopy", as we call it, is unique in that it uses statistical methods to provide direct measurements of these forces at the individual particle scale, while still being sufficiently high-throughput to produce meaningful population level data. In this talk I will introduce the technology, it's advantages, and some of the major uses. Specific case studies will include label-free monitoring of binding of individual antibodies onto single viruses and the measurement of the strength of nanoparticle coatings used for steric stabilization.
Carqueijeiro, I; Masini, E; Foureau, E; Sepúlveda, L J; Marais, E; Lanoue, A; Besseau, S; Papon, N; Clastre, M; Dugé de Bernonville, T; Glévarec, G; Atehortùa, L; Oudin, A; Courdavault, V
Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.
Full Text Available From October 2014 to March 2015, French Polynesia experienced for the first time a chikungunya outbreak. Two Aedes mosquitoes may have contributed to chikungunya virus (CHIKV transmission in French Polynesia: the worldwide distributed Ae. aegypti and the Polynesian islands-endemic Ae. polynesiensis mosquito.To investigate the vector competence of French Polynesian populations of Ae. aegypti and Ae. polynesiensis for CHIKV, mosquitoes were exposed per os at viral titers of 7 logs tissue culture infectious dose 50%. At 2, 6, 9, 14 and 21 days post-infection (dpi, saliva was collected from each mosquito and inoculated onto C6/36 mosquito cells to check for the presence of CHIKV infectious particles. Legs and body (thorax and abdomen of each mosquito were also collected at the different dpi and submitted separately to viral RNA extraction and CHIKV real-time RT-PCR.CHIKV infection rate, dissemination and transmission efficiencies ranged from 7-90%, 18-78% and 5-53% respectively for Ae. aegypti and from 39-41%, 3-17% and 0-14% respectively for Ae. polynesiensis, depending on the dpi. Infectious saliva was found as early as 2 dpi for Ae. aegypti and from 6 dpi for Ae. polynesiensis. Our laboratory results confirm that the French Polynesian population of Ae. aegypti is highly competent for CHIKV and they provide clear evidence for Ae. polynesiensis to act as an efficient CHIKV vector.As supported by our findings, the presence of two CHIKV competent vectors in French Polynesia certainly contributed to enabling this virus to quickly disseminate from the urban/peri-urban areas colonized by Ae. aegypti to the most remote atolls where Ae. polynesiensis is predominating. Ae. polynesiensis was probably involved in the recent chikungunya outbreaks in Samoa and the Cook Islands. Moreover, this vector may contribute to the risk for CHIKV to emerge in other Polynesian islands like Fiji, and more particularly Wallis where there is no Ae. aegypti.
Michael R. Strand
Full Text Available Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1 they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2 they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution.
Wang, Dai; Phan, Shannon; DiStefano, Daniel J; Citron, Michael P; Callahan, Cheryl L; Indrawati, Lani; Dubey, Sheri A; Heidecker, Gwendolyn J; Govindarajan, Dhanasekaran; Liang, Xiaoping; He, Biao; Espeseth, Amy S
Human respiratory syncytial virus (RSV) is a common cause of severe respiratory disease among infants, immunocompromised individuals, and the elderly. No licensed vaccine is currently available. In this study, we evaluated two parainfluenza virus 5 (PIV5)-vectored vaccines expressing RSV F (PIV5/F) or G (PIV5/G) protein in the cotton rat and African green monkey models for their replication, immunogenicity, and efficacy of protection against RSV challenge. Following a single intranasal inoculation, both animal species shed the vaccine viruses for a limited time but without noticeable clinical symptoms. In cotton rats, the vaccines elicited RSV F- or G-specific serum antibodies and conferred complete lung protection against RSV challenge at doses as low as 103 PFU. Neither vaccine produced the enhanced lung pathology observed in animals immunized with formalin-inactivated RSV. In African green monkeys, vaccine-induced serum and mucosal antibody responses were readily detected, as well. PIV5/F provided nearly complete protection against RSV infection in the upper and lower respiratory tract at a dose of 106 PFU of vaccine. At the same dose levels, PIV5/G was less efficacious. Both PIV5/F and PIV5/G were also able to boost neutralization titers in RSV-preexposed African green monkeys. Overall, our data indicated that PIV5/F is a promising RSV vaccine candidate.IMPORTANCE A safe and efficacious respiratory syncytial virus (RSV) vaccine remains elusive. We tested the recombinant parainfluenza virus 5 (PIV5) vectors expressing RSV glycoproteins for their immunogenicity and protective efficacy in cotton rats and African green monkeys, which are among the best available animal models to study RSV infection. In both species, a single dose of intranasal immunization with PIV5-vectored vaccines was able to produce systemic and local immunity and to protect animals from RSV challenge. The vaccines could also boost RSV neutralization antibody titers in African green monkeys
Deardorff, Eleanor R.; Estrada-Franco, Jose G.; Freier, Jerome E.; Navarro-Lopez, Roberto; Da Rosa, Amelia Travassos; Tesh, Robert B.; Weaver, Scott C.
Enzootic Venezuelan equine encephalitis virus (VEEV) has been known to occur in Mexico since the 1960s. The first natural equine epizootic was recognized in Chiapas in 1993 and since then, numerous studies have characterized the etiologic strains, including reverse genetic studies that incriminated a specific mutation that enhanced infection of epizootic mosquito vectors. The aim of this study was to determine the mosquito and rodent species involved in enzootic maintenance of subtype IE VEEV in coastal Chiapas. A longitudinal study was conducted over a year to discern which species and habitats could be associated with VEEV circulation. Antibody was rarely detected in mammals and virus was not isolated from mosquitoes. Additionally, Culex (Melanoconion) taeniopus populations were found to be spatially related to high levels of human and bovine seroprevalence. These mosquito populations were concentrated in areas that appear to represent foci of stable, enzootic VEEV circulation. PMID:22144461
Yin, Jiechao; Ren, Xiaofeng; Tian, Zhijun; Li, Yijing
Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed.
Eliasson, Dubravka Grdic; Helgeby, Anja; Schön, Karin; Nygren, Caroline; El-Bakkouri, Karim; Fiers, Walter; Saelens, Xavier; Lövgren, Karin Bengtsson; Nyström, Ida; Lycke, Nils Y
Here we demonstrate that by using non-toxic fractions of saponin combined with CTA1-DD we can achieve a safe and above all highly efficacious mucosal adjuvant vector. We optimized the construction, tested the requirements for function and evaluated proof-of-concept in an influenza A virus challenge model. We demonstrated that the CTA1-3M2e-DD/ISCOMS vector provided 100% protection against mortality and greatly reduced morbidity in the mouse model. The immunogenicity of the vector was superior to other vaccine formulations using the ISCOM or CTA1-DD adjuvants alone. The versatility of the vector was best exemplified by the many options to insert, incorporate or admix vaccine antigens with the vector. Furthermore, the CTA1-3M2e-DD/ISCOMS could be kept 1 year at 4°C or as a freeze-dried powder without affecting immunogenicity or adjuvanticity of the vector. Strong serum IgG and mucosal IgA responses were elicited and CD4 T cell responses were greatly enhanced after intranasal administration of the combined vector. Together these findings hold promise for the combined vector as a mucosal vaccine against influenza virus infections including pandemic influenza. The CTA1-DD/ISCOMS technology represents a breakthrough in mucosal vaccine vector design which successfully combines immunomodulation and targeting in a safe and stable particulate formation. Copyright © 2011 Elsevier Ltd. All rights reserved.
Ball, L A
RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.
Chinnaraja, C; Viswanathan, R
Yellow leaf caused by Sugarcane yellow leaf virus (SCYLV) is a serious constraint to sugarcane production in India and currently the disease epidemics occur on many of the susceptible varieties under field conditions. Studies were conducted on the virus transmission by sugarcane aphid Melanaphis sacchari in sugarcane by inoculating virus-free meristem derived from micro- propagated plants of sugarcane cv Co 86032 with viruliferous aphids. Virus transmission was confirmed through RT-PCR assays and subsequently SCYLV population was established through RT-qPCR. A maximum of 22.3 × 10(3), 3.16 × 10(6) and 4.78 × 10(6) copies of SCYLV-RNA targets were recorded in the plants after 7, 180 and 300 days, respectively. This study showed that the aphid species M. sacchari acts as an effective vector of SCYLV. The relative standard curve method in RT-qPCR efficiently detected the increment in SCYLV copy numbers in sugarcane following transmission through M. sacchari.
Aspden, Kate; Passmore, Jo-Ann; Tiedt, Friedrich; Williamson, Anna-Lise
Lumpy skin disease virus (LSDV), a capripoxvirus with a host range limited to ruminants, was evaluated as a replication-deficient vaccine vector for use in non-ruminant hosts. By using the rabies virus glycoprotein (RG) as a model antigen, it was demonstrated that recombinant LSDV encoding the rabies glycoprotein (rLSDV-RG) was able to express RG in both permissive (ruminant) and non-permissive (non-ruminant) cells. The recombinant LSDV, however, replicated to maturity only in permissive but not in non-permissive cells. Recombinant LSDV-RG was assessed for its ability to generate immunity against RG in non-ruminant hosts (rabbits and mice). Rabbits inoculated with rLSDV-RG produced rabies virus (RV) neutralizing antibodies at levels twofold higher than those reported by the WHO to be protective. BALB/c mice immunized with rLSDV-RG elicited levels of RV-specific cellular immunity (T-cell proliferation) comparable with those of mice immunized with a commercial inactivated rabies vaccine (Verorab; Pasteur Merieux). Most importantly, mice immunized with rLSDV-RG were protected from an aggressive intracranial rabies virus challenge.
Boye, Sanford L.; Bennett, Antonette; Scalabrino, Miranda L.; McCullough, K. Tyler; Van Vliet, Kim; Choudhury, Shreyasi; Ruan, Qing; Peterson, James
ABSTRACT Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors
Full Text Available Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV, a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg RNA which is also required as bicistronic mRNA for the capsid (core protein and the reverse transcriptase (Pol; their open reading frames (ORFs overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES. We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR and humanized Renilla green fluorescent protein (hrGFP produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to
Chen, Qiang; He, Junyun; Phoolcharoen, Waranyoo; Mason, Hugh S
Expression of recombinant vaccine antigens and monoclonal antibodies using plant viral vectors has developed extensively during the past several years. The approach benefits from high yields of recombinant protein obtained within days after transient delivery of viral vectors to leaves of Nicotiana benthamiana, a tobacco relative. Modified viral genomes of both RNA and DNA viruses have been created. Geminiviruses such as bean yellow dwarf virus (BeYDV) have a small, single stranded DNA genome that replicates in the nucleus of an infected plant cell, using the cellular DNA synthesis apparatus and a virus-encoded replication initiator protein (Rep). BeYDV-derived expression vectors contain deletions of the viral genes encoding coat and movement proteins and insertion of an expression cassette for a protein of interest. Delivery of the geminiviral vector to leaf cells via Agrobacterium-mediated delivery produces very high levels of recombinant DNA that can act as a transcription template, yielding high levels of mRNA for the protein of interest. Several vaccine antigens, including Norwalk virus capsid protein and hepatitis B core antigen, were expressed using the BeYDV vector at levels up to 1 mg per g of leaf mass. BeYDV replicons can be stacked in the same vector molecule by linking them in tandem, which enables production of multi-subunit proteins like monoclonal antibody (mAb) heavy and light chains. The protective mAb 6D8 against Ebola virus was produced at 0.5 mg per g of leaf mass. Multi-replicon vectors could be conveniently used to produce protein complexes, e.g. virus-like particles that require two or more subunits.
Wang, Zihua; Wu, Li; Cheng, Xin; Liu, Shizhu; Li, Baosheng; Li, Haijun; Kang, Fubiao; Wang, Junping; Xia, Huan; Ping, Caiyan; Nassal, Michael; Sun, Dianxing
Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better
Linke, Lyndsey M; Wilusz, Jeffrey; Pabilonia, Kristy L; Fruehauf, Johannes; Magnuson, Roberta; Olea-Popelka, Francisco; Triantis, Joni; Landolt, Gabriele; Salman, Mo
Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.
Brian B. Gowen
Full Text Available Rift Valley fever virus (RVFV causes severe disease in humans and ungulates. The virus can be transmitted by mosquitoes, direct contact with infected tissues or fluids, or aerosol, making it a significant biological threat for which there is no approved vaccine or therapeutic. Herein we describe the evaluation of DEF201, an adenovirus-vectored interferon alpha which addresses the limitations of recombinant interferon alpha protein (cost, short half-life, as a pre- and post-exposure treatment in a lethal hamster RVFV challenge model. DEF201 was delivered intranasally to stimulate mucosal immunity and effectively bypass any pre-existing immunity to the vector. Complete protection against RVFV infection was observed from a single dose of DEF201 administered one or seven days prior to challenge while all control animals succumbed within three days of infection. Efficacy of treatment administered two weeks prior to challenge was limited. Post‑exposure, DEF201 was able to confer significant protection when dosed at 30 min or 6 h, but not at 24 h post-RVFV challenge. Protection was associated with reductions in serum and tissue viral loads. Our findings suggest that DEF201 may be a useful countermeasure against RVFV infection and further demonstrates its broad-spectrum capacity to stimulate single dose protective immunity.
Maize fine streak virus (MFSV) is an emerging virus of maize that is transmitted by an insect vector, the leafhopper called Graminella nigrifrons. Virus transmission by the leafhopper requires that the virus enter into and multiply in insect cells, tissues and organs before being transmitted to a ne...
Full Text Available In 2013-2014, French Polynesia experienced for the first time a Zika outbreak. Two Aedes mosquitoes may have contributed to Zika virus (ZIKV transmission in French Polynesia: the worldwide distributed Ae. aegypti and the Polynesian islands-endemic Ae. polynesiensis mosquito.To evaluate their vector competence for ZIKV, mosquitoes were infected per os at viral titers of 7 logs tissue culture infectious dose 50%. At several days post-infection (dpi, saliva was collected from each mosquito and inoculated onto C6/36 mosquito cells to check for the presence of ZIKV infectious particles. Legs and body of each mosquito were also collected and submitted separately to RNA extraction and ZIKV RT-PCR. In Ae. aegypti the infection rate was high as early as 6 dpi and the dissemination efficiency get substantial from 9 dpi while the both rates remained quite low in Ae. polynesiensis. The transmission efficiency was poor in Ae. aegypti until 14 dpi and no infectious saliva was found in Ae. polynesiensis at the time points studied.In our experimental conditions, the late ability of the French Polynesian Ae. aegypti to transmit ZIKV added by the poor competence of Ae. polynesiensis for this virus suggest the possible contribution of another vector for the propagation of ZIKV during the outbreak, in particular in remote islands where Ae. polynesiensis is predominating.
Es-Sette, Nargys; Ajaoud, Malika; Bichaud, Laurence; Hamdi, Salsabil; Mellouki, Fouad; Charrel, Rémi N; Lemrani, Meryem
An entomological study using CDC miniature light-traps was performed in El Hanchane locality, where cutaneous leishmaniasis (CL) was emerging during the summer of 2011. The aim of this study is to identify the vectors of Leishmania and of phleboviruses. In the field, a total of 643 sandfly specimens were collected, identified by morphological keys and categorized by sex and species. A total of nine distinct species were morphologically identified where seven belonged to the Phlebotomus genus and two species to the Sergentomyia genus. Phlebotomus sergenti was the most abundant species (76%). Phleboviruses were detected by nested RT-PCR using 30 pooled sandflies while P. sergenti females were tested individually for infections of Leishmania species. By using ITS1-PCR-RFLP approach, Leishmania tropica DNA was detected in 10 females, caught in this emerging focus, and provide additional evidence in favour of the role of P. sergenti as vector of L. tropica in Morocco. Real-time PCR screening for phlebovirus RNA, using an assay targeting the polymerase gene, showed positive result in one pool of male P. sergenti. In this study, P. sergenti were infected by L. tropica and Toscana virus. To our knowledge, actually this is the first time that Toscana virus has been detected in P. sergenti.
Full Text Available The 2015-16 Zika virus pandemic originating in Latin America led to predictions of a catastrophic global spread of the disease. Since the current outbreak began in Brazil in May 2015 local transmission of Zika has been reported in over 60 countries and territories, with over 750 thousand confirmed and suspected cases. As a result of its range expansion attention has focused on possible modes of transmission, of which the arthropod vector-based disease spread cycle involving Aedes species is believed to be the most important. Additional causes of concern are the emerging new links between Zika disease and Guillain-Barre Syndrome (GBS, and a once rare congenital disease, microcephaly.Like dengue and chikungunya, the geographic establishment of Zika is thought to be limited by the occurrence of its principal vector mosquito species, Ae. aegypti and, possibly, Ae. albopictus. While Ae. albopictus populations are more widely established than those of Ae. aegypti, the relative competence of these species as a Zika vector is unknown. The analysis reported here presents a global risk model that considers the role of each vector species independently, and quantifies the potential spreading risk of Zika into new regions. Six scenarios are evaluated which vary in the weight assigned to Ae. albopictus as a possible spreading vector. The scenarios are bounded by the extreme assumptions that spread is driven by air travel and Ae. aegypti presence alone and spread driven equally by both species. For each scenario destination cities at highest risk of Zika outbreaks are prioritized, as are source cities in affected regions. Finally, intercontinental air travel routes that pose the highest risk for Zika spread are also ranked. The results are compared between scenarios.Results from the analysis reveal that if Ae. aegypti is the only competent Zika vector, then risk is geographically limited; in North America mainly to Florida and Texas. However, if Ae
Mason, Giovanna; Roggero, Piero; Tavella, Luciana
A method for rapid and reliable detection of Tomato spotted wilt virus (TSWV) (Tospovirus, Bunyaviridae) in its vector Frankliniella occidentalis (Thysanoptera Thripidae) would be a useful tool for studying the epidemiology of this virus. A RT-PCR method developed for this purpose is reported. The method was tested on thrips involved in laboratory transmission trials and on thrips collected in the field, whose capability to transmit TSWV was checked previously by leaf disk assays. The RT-PCR results were consistent with the results obtained by the leaf disk assays. Among thrips involved in laboratory experiments, 97% of the adults that transmitted TSWV were positive by RT-PCR; as did some non-transmitter adults reacted, whereas among field-collected thrips only the individuals able to transmit were positive by RT-PCR. In addition, healthy thrips were allowed to feed as adults on virus-infected leaves for 48 h, and then examined by RT-PCR immediately or after starving or feeding on virus-free plants for various times, to determine if virus ingested (but not transmissible) was also detectable. The virus was detectable immediately after the feed or within 12 and 24 h for individuals starved or fed on virus-free plants, respectively, but not after those periods. Thus, the method could detect rapidly and reliably the virus in vectors from the field, providing 24 h of starving to avoid positive RT-PCR results from thrips simply carrying the virus.
Soltész, Zoltán; Erdélyi, Károly; Bakonyi, Tamás; Barna, Mónika; Szentpáli-Gavallér, Katalin; Solt, Szabolcs; Horváth, Éva; Palatitz, Péter; Kotymán, László; Dán, Ádám; Papp, László; Harnos, Andrea; Fehérvári, Péter
Avian host species have different roles in the amplification and maintenance of West Nile virus (WNV), therefore identifying key taxa is vital in understanding WNV epidemics. Here, we present a comprehensive case study conducted on red-footed falcons, where host-vector, vector-virus and host-virus interactions were simultaneously studied to evaluate host species contribution to WNV circulation qualitatively. Mosquitoes were trapped inside red-footed falcon nest-boxes by a method originally developed for the capture of blackflies and midges. We showed that this approach is also efficient for trapping mosquitoes and that the number of trapped vectors is a function of host attraction. Brood size and nestling age had a positive effect on the number of attracted Culex pipiens individuals while the blood-feeding success rate of both dominant Culex species (Culex pipiens and Culex modestus) markedly decreased after the nestlings reached 14 days of age. Using RT-PCR, we showed that WNV was present in these mosquitoes with 4.2% (CI: 0.9-7.5%) prevalence. We did not detect WNV in any of the nestling blood samples. However, a relatively high seroprevalence (25.4% CI: 18.8-33.2%) was detected with an enzyme-linked immunoabsorbent assay (ELISA). Using the ELISA OD ratios as a proxy to antibody titers, we showed that older seropositive nestlings have lower antibody levels than their younger conspecifics and that hatching order negatively influences antibody levels in broods with seropositive nestlings. Red-footed falcons in the studied system are exposed to a local sylvatic WNV circulation, and the risk of infection is higher for younger nestlings. However, the lack of individuals with viremia and the high WNV seroprevalence, indicate that either host has a very short viremic period or that a large percentage of nestlings in the population receive maternal antibodies. This latter assumption is supported by the age and hatching order dependence of antibody levels found for
Macharia, Isaac; Backhouse, David; Skilton, Rob; Ateka, Elijah; Wu, Shu-Biao; Njahira, Moses; Maina, Solomon; Harvey, Jagger
Thrips have been recognized as primary vectors of tomato spotted wilt virus (TSWV) with Frankliniella occidentalis (Pergande) reported as the most important and efficient vector, while other species such as Thrips tabaci Lindeman also include populations that can vector the virus. A study was undertaken to establish the diversity of thrips and presence of vectors for TSWV in four major tomato production areas in Kenya. The cytochrome oxidase 1 (CO1) gene was used to generate sequences from thrips samples collected from tomatoes and weeds, and phylogenetic analysis done to establish the variation within potential vector populations. Ceratothripoides brunneus Bagnall was the predominant species of thrips in all areas. F. occidentalis and T. tabaci were abundant in Nakuru, Kirinyaga, and Loitokitok but not detected at Bungoma. Other vectors of tospoviruses identified in low numbers were Frankliniella schultzei (Trybom) and Scirtothrips dorsalis Hood. Variation was observed in T. tabaci, F. occidentalis, and F. schultzei. Kenyan specimens of T. tabaci from tomato belonged to the arrhenotokous group, while those of F. occidentalis clustered with the Western flower thrips G group. The detection of RNA of TSWV in both of these species of thrips supported the role they play as vectors. The study has demonstrated the high diversity of thrips species in tomato production and the occurrence of important vectors of TSWV and other tospoviruses. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: email@example.com.
Pinheiro, Patricia V; Ghanim, Murad; Alexander, Mariko; Rebelo, Ana Rita; Santos, Rogerio S; Orsburn, Benjamin C; Gray, Stewart; Cilia, Michelle
The green peach aphid, Myzus persicae , is a vector of the Potato leafroll virus (PLRV, Luteoviridae), transmitted exclusively by aphids in a circulative manner. PLRV transmission efficiency was significantly reduced when a clonal lineage of M. persicae was reared on turnip as compared with the weed physalis, and this was a transient effect caused by a host-switch response. A trend of higher PLRV titer in physalis-reared aphids as compared with turnip-reared aphids was observed at 24 h and 72 h after virus acquisition. The major difference in the proteomes of these aphids was the up-regulation of predicted lysosomal enzymes, in particular the cysteine protease cathepsin B (cathB), in aphids reared on turnip. The aphid midgut is the site of PLRV acquisition, and cathB and PLRV localization were starkly different in midguts of the aphids reared on the two host plants. In viruliferous aphids that were reared on turnip, there was near complete colocalization of cathB and PLRV at the cell membranes, which was not observed in physalis-reared aphids. Chemical inhibition of cathB restored the ability of aphids reared on turnip to transmit PLRV in a dose-dependent manner, showing that the increased activity of cathB and other cysteine proteases at the cell membrane indirectly decreased virus transmission by aphids. Understanding how the host plant influences virus transmission by aphids is critical for growers to manage the spread of virus among field crops. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Quetglas, J I; Fioravanti, J; Ardaiz, N; Medina-Echeverz, J; Baraibar, I; Prieto, J; Smerdou, C; Berraondo, P
Semliki Forest virus (SFV) represents a promising gene therapy vector for tumor treatment, because it produces high levels of recombinant therapeutic proteins while inducing apoptosis in infected cells. In this study, we constructed a SFV vector expressing murine interferon alpha (IFNα). IFNα displays antitumor activity mainly by enhancing an antitumor immune response, as well as by a direct antiproliferative effect. In spite of the antiviral activity of IFNα, SFV-IFN could be produced in BHK cells at high titers. This vector was able to infect TC-1 cells, a tumor cell line expressing E6 and E7 proteins of human papillomavirus, leading to high production of IFNα both in vitro and in vivo. When injected into subcutaneous TC-1 tumors implanted in mice, SFV-IFN was able to induce an E7-specific cytotoxic T lymphocyte response, and to modify tumor infiltrating immune cells, reducing the percentage of T regulatory cells and activating myeloid cells. As a consequence, SFV-IFN was able to eradicate 58% of established tumors treated 21 days after implantation with long-term tumor-free survival and very low toxicity. SFV-IFN was also able to induce significant antitumor responses in a subcutaneous tumor model of murine colon adenocarcimoma. These data suggest that local production of IFNα by intratumoral injection of recombinant SFV-IFN could represent a potent new strategy to treat tumors in patients.
Malachy I. Okeke
Full Text Available Modified vaccinia virus Ankara (MVA is the vector of choice for human and veterinary applications due to its strong safety profile and immunogenicity in vivo. The use of MVA and MVA-vectored vaccines against human and animal diseases must comply with regulatory requirements as they pertain to environmental risk assessment, particularly the characterization of potential adverse effects to humans, animals and the environment. MVA and recombinant MVA are widely believed to pose low or negligible risk to ecosystem health. However, key aspects of MVA biology require further research in order to provide data needed to evaluate the potential risks that may occur due to the use of MVA and MVA-vectored vaccines. The purpose of this paper is to identify knowledge gaps in the biology of MVA and recombinant MVA that are of relevance to its hazard characterization and discuss ongoing and future experiments aimed at providing data necessary to fill in the knowledge gaps. In addition, we presented arguments for the inclusion of uncertainty analysis and experimental investigation of verifiable worst-case scenarios in the environmental risk assessment of MVA and recombinant MVA. These will contribute to improved risk assessment of MVA and recombinant MVA vaccines.
Gossner, Céline M; Marrama, Laurence; Carson, Marianne; Allerberger, Franz; Calistri, Paolo; Dilaveris, Dimitrios; Lecollinet, Sylvie; Morgan, Dilys; Nowotny, Norbert; Paty, Marie-Claire; Pervanidou, Danai; Rizzo, Caterina; Roberts, Helen; Schmoll, Friedrich; Van Bortel, Wim; Gervelmeyer, Andrea
This article uses the experience of five European countries to review the integrated approaches (human, animal and vector) for surveillance and monitoring of West Nile virus (WNV) at national and European levels. The epidemiological situation of West Nile fever in Europe is heterogeneous. No model of surveillance and monitoring fits all, hence this article merely encourages countries to implement the integrated approach that meets their needs. Integration of surveillance and monitoring activities conducted by the public health authorities, the animal health authorities and the authorities in charge of vector surveillance and control should improve efficiency and save resources by implementing targeted measures. The creation of a formal interagency working group is identified as a crucial step towards integration. Blood safety is a key incentive for public health authorities to allocate sufficient resources for WNV surveillance, while the facts that an effective vaccine is available for horses and that most infected animals remain asymptomatic make the disease a lesser priority for animal health authorities. The examples described here can support other European countries wishing to strengthen their WNV surveillance or preparedness, and also serve as a model for surveillance and monitoring of other (vector-borne) zoonotic infections. This article is copyright of The Authors, 2017.
Full Text Available Boston, Massachusetts, was the site of the 9th International Conference on Oncolytic Virus Therapeutics held 13–16 June 2015. An overarching theme of the meeting was the continued development of combinatorial treatment regimens to bolster the therapeutic potential of oncolytic viruses (OVs. Several talks focused on combining OVs with immune checkpoint inhibitors in a wide array of tumors, signaling an experimental and thematic shift toward driving immune activation to clear a tumor versus relying on direct viral oncolysis. An important aspect of the meeting was the variety of ongoing OV clinical trials. Topics ranged from basic virology to clinical trials and from academic research to intellectual property and biotechnology. There was much excitement due to the US Food and Drug Administration’s recent consideration of talimogene laherparepvec (T-VEC for the treatment of advanced melanoma (T-VEC was approved in October, following the conference. Here, we summarize the meeting’s primary themes, which reflect the current state of the field.
Full Text Available Efforts to reduce the viral load of human immunodeficiency virus type 1 (HIV-1 during long-term treatment are challenged by the evolution of anti-viral resistance mutants. Recent studies have shown that gene therapy approaches based on conditionally replicating vectors (CRVs could have many advantages over anti-viral drugs and other approaches to therapy, potentially including the ability to circumvent the problem of evolved resistance. However, research to date has not explored the evolutionary consequences of long-term treatment of HIV-1 infections with conditionally replicating vectors. In this study, we analyze a computational model of the within-host co-evolutionary dynamics of HIV-1 and conditionally replicating vectors, using the recently proposed 'therapeutic interfering particle' as an example. The model keeps track of the stochastic process of viral mutation, and the deterministic population dynamics of T cells as well as different strains of CRV and HIV-1 particles. We show that early in the co-infection, mutant HIV-1 genotypes that escape suppression by CRV therapy appear; this is similar to the dynamics observed in drug treatments and other gene therapies. In contrast to other treatments, however, the CRV population is able to evolve and catch up with the dominant HIV-1 escape mutant and persist long-term in most cases. On evolutionary grounds, gene therapies based on CRVs appear to be a promising tool for long-term treatment of HIV-1. Our model allows us to propose design principles to optimize the efficacy of this class of gene therapies. In addition, because of the analogy between CRVs and naturally-occurring defective interfering particles, our results also shed light on the co-evolutionary dynamics of wild-type viruses and their defective interfering particles during natural infections.
Noriko eYamagishi; Chunjiang eLi; Nobuyuki eYoshikawa
Plant viral vectors are superior tools for genetic manipulation, allowing rapid induction or suppression of expression of a target gene in plants. This is a particularly effective technology for use in breeding fruit trees, which are difficult to manipulate using recombinant DNA technologies. We reported previously that if apple seed embryos (cotyledons) are infected with an Apple latent spherical virus (ALSV) vector (ALSV-AtFT/MdTFL1) concurrently expressing the Arabidopsis thaliana florigen...
Liu, Xiaohui; Yang, Youtian; Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F; Niu, Xuefeng; Guo, Xiaofeng
The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines.
Vu, Hiep L X; Ma, Fangrui; Laegreid, William W; Pattnaik, Asit K; Steffen, David; Doster, Alan R; Osorio, Fernando A
Current vaccines do not provide sufficient levels of protection against divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains circulating in the field, mainly due to the substantial variation of the viral genome. We describe here a novel approach to generate a PRRSV vaccine candidate that could confer unprecedented levels of heterologous protection against divergent PRRSV isolates. By using a set of 59 nonredundant, full-genome sequences of type 2 PRRSVs, a consensus genome (designated PRRSV-CON) was generated by aligning these 59 PRRSV full-genome sequences, followed by selecting the most common nucleotide found at each position of the alignment. Next, the synthetic PRRSV-CON strain was generated through the use of reverse genetics. PRRSV-CON replicates as efficiently as our prototype PRRSV strain FL12, both in vitro and in vivo. Importantly, when inoculated into pigs, PRRSV-CON confers significantly broader levels of heterologous protection than does wild-type PRRSV. Collectively, our data demonstrate that PRRSV-CON can serve as an excellent candidate for the development of a broadly protective PRRSV vaccine. The extraordinary genetic variation of RNA viruses poses a monumental challenge for the development of broadly protective vaccines against these viruses. To minimize the genetic dissimilarity between vaccine immunogens and contemporary circulating viruses, computational strategies have been developed for the generation of artificial immunogen sequences (so-called "centralized" sequences) that have equal genetic distances to the circulating viruses. Thus far, the generation of centralized vaccine immunogens has been carried out at the level of individual viral proteins. We expand this concept to PRRSV, a highly variable RNA virus, by creating a synthetic PRRSV strain based on a centralized PRRSV genome sequence. This study provides the first example of centralizing the whole genome of an RNA virus to improve vaccine coverage. This
Grischott, Franca; Puhan, Milo; Hatz, Christoph; Schlagenhauf, Patricia
Increasing numbers of confirmed cases of Zika virus (ZIKV) infection resulting from non-mosquito-borne transmission have been reported. We performed a systematic literature review (PRISMA guidelines) on intrauterine, intrapartum, sexual and animal bite ZIKV transmission. The presence of the virus in breast milk, urine, saliva and blood transfusions was also reviewed. The search resulted in 285 papers of possible relevance, of which we included 53 in the systematic review. Mother-to-child transmission was most frequently described with adverse infant outcomes including microcephaly, intracranial calcification and fetal death. Zika virus RNA has been detected in amniotic fluid, breast milk, seminal fluid, saliva, urine and blood. Semen and blood products have proved to be infectious. Male-to-female and male-to-male ZIKV transmission is documented. There are contradictory results concerning the infectiousness of breast milk and urine and data on saliva, animal bites, transplantation, needlestick injury and laboratory work are inconclusive. Our systematic analysis shows that non-vector-borne ZIKV transmission plays a role in the spread of ZIKV and has great societal impact. It has important public health implications for the prevention and control of ZIKV globally and will be a basis for policy and further research. Copyright © 2016 Elsevier Ltd. All rights reserved.
Full Text Available Aphids are the most important vectors of potato viruses during the crop’s growing season. The most widespread and damaging viruses, the potato virus Y and potato leaf roll virus, are transmitted by aphids in non-persistent and persistent manner, respectively. The two viruses cause the greatest concern of potato producers and a great constraint to seed potato production in Serbia, the region and across the world. Potato virus Y is particularly harmful, given its distribution and spreading rate. Seed potato production systems under well-managed conditions involve a series of virus control measures, including the monitoring of outbreaks of winged aphids, their abundance and species composition, in order to forecast virosis, i.e. potential plant and tuber infection periods. Monitoring the aphid vectors of potato viruses enables determination of optimum dates for haulm destruction when higher than normal numbers of winged aphids as vectors of economically harmful diseases have been observed. Haulm destruction in a potato crop reduces the risk of plant infection and virus translocation from the aboveground parts to tubers, thus keeping the proportion of infected tubers within tolerance limits allowed for certain categories of seed potatoes. This practice has positive effects if used in combination with other viral disease control measures; otherwise, it becomes ineffective. This paper provides an integral analysis of the effects and role of monitoring outbreaks of aphids, their abundance and species composition in timing haulm growth termination to prevent plant infection, virus translocation and tuber infestation in potato crops in Serbia and the wider region.
Keough, Stacy; Han, Jinlong; Shuman, Tyler; Wise, Kiersten; Nachappa, Punya
Soybean vein necrosis virus (SVNV) is an emerging Tospovirus that is now considered to be the most widespread soybean virus in the United States. SVNV is transmitted from plant-to-plant by soybean thrips, Neohydatothrips variabilis (Beach). We hypothesized that a positive interaction between the host plant, SVNV, and the vector may have resulted in the widespread distribution of the virus in a short span of time. Our study found that SVNV-infected N. variabilis females produced significantly more offspring compared with non-infected females. No other life-history trait varied between SVNV-infected and non-infected thrips. There was considerable variation in SVNV copy number in infected thrips ranging from 10(2) -10(6) Moreover, there was a significant negative correlation between SVNV copy number and fecundity in infected N. variabilis This suggests that excessive virus accumulation may result in lower viability of N. variabilis In choice tests, SVNV-infected N. variabilis preferred to feed on non-infected leaflets compared with infected leaflets. Vector competence assays indicated that Frankliniella tritici and Frankliniella fusca can transmit SVNV, but at a lower efficiency than N. variabilis Comparison of life history of between the primary and secondary vectors showed that N. variabilis had the highest fecundity, but F. tritici had the shortest development time and greatest larval survival. Taken together, the increased fecundity of SVNV-infected N. variabilis, their apparent preference for non-infected host plants, in conjunction with the ability of secondary vectors to survive and reproduce on soybean may, in part, explain the rapid spread of SVNV in the United States. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: firstname.lastname@example.org.
Full Text Available Abstract Background West Nile Virus (WNV transmission in Italy was first reported in 1998 as an equine outbreak near the swamps of Padule di Fucecchio, Tuscany. No other cases were identified during the following decade until 2008, when horse and human outbreaks were reported in Emilia Romagna, North Italy. Since then, WNV outbreaks have occurred annually, spreading from their initial northern foci throughout the country. Following the outbreak in 1998 the Italian public health authority defined a surveillance plan to detect WNV circulation in birds, horses and mosquitoes. By applying spatial statistical analysis (spatial point pattern analysis and models (Bayesian GLMM models to a longitudinal dataset on the abundance of the three putative WNV vectors [Ochlerotatus caspius (Pallas 1771, Culex pipiens (Linnaeus 1758 and Culex modestus (Ficalbi 1890] in eastern Piedmont, we quantified their abundance and distribution in space and time and generated prediction maps outlining the areas with the highest vector productivity and potential for WNV introduction and amplification. Results The highest abundance and significant spatial clusters of Oc. caspius and Cx. modestus were in proximity to rice fields, and for Cx. pipiens, in proximity to highly populated urban areas. The GLMM model showed the importance of weather conditions and environmental factors in predicting mosquito abundance. Distance from the preferential breeding sites and elevation were negatively associated with the number of collected mosquitoes. The Normalized Difference Vegetation Index (NDVI was positively correlated with mosquito abundance in rice fields (Oc. caspius and Cx. modestus. Based on the best models, we developed prediction maps for the year 2010 outlining the areas where high abundance of vectors could favour the introduction and amplification of WNV. Conclusions Our findings provide useful information for surveillance activities aiming to identify locations where the
Vida L. Hodara; Velasquillo, M. Cristina; Parodi, Laura M.; Giavedoni, Luis D.
Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that...
Sonja Hall-Mendelin; Alyssa T. Pyke; Moore, Peter R.; Mackay, Ian M.; Jamie L McMahon; Scott A. Ritchie; Carmel T. Taylor; Moore, Frederick A.J.; van den Hurk, Andrew F.
Background Within the last 10 years Zika virus (ZIKV) has caused unprecedented epidemics of human disease in the nations and territories of the western Pacific and South America, and continues to escalate in both endemic and non-endemic regions. We evaluated the vector competence of Australian mosquitoes for ZIKV to assess their potential role in virus transmission. Methodology/Principal Findings Mosquitoes were exposed to infectious blood meals containing the prototype African ZIKV strain. A...
Métais, J-Y; Topp, S; Doty, R T; Borate, B; Nguyen, A-D; Wolfsberg, T G; Abkowitz, J L; Dunbar, C E
Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.
VAN DER Saag, M R; Ward, M P; Kirkland, P D
Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a number of globally important arboviruses that affect livestock, including bluetongue virus (BTV), African horse sickness virus and the recently emerged Schmallenberg virus. In this study, a model using embryonated chicken eggs (ECEs) was utilized to undertake vector competence studies of Australian Culicoides spp. for 13 laboratory-adapted or wild-type virus strains of BTV. A total of 7393 Culicoides brevitarsis were reared from bovine dung, and 3364 Culicoides were induced to feed from ECEs infected with different strains of BTV. Of those, 911 (27%) survived the putative extrinsic incubation period of 9-12 days. In some trials, virus was also transmitted onward to uninfected ECEs, completing the transmission cycle. This model does not rely on the use of colonized midges and has the capacity to assess the vector competence of field-collected insects with strains of virus that have not previously been passaged in laboratory culture systems. There is also potential for this model to be used in investigations of the competence of Culicoides spp. for other arboviruses. © 2017 The Royal Entomological Society.
Ko, Eun-Ju; Kwon, Young-Man; Lee, Jong Seok; Hwang, Hye Suk; Yoo, Si-Eun; Lee, Yu-Na; Lee, Young-Tae; Kim, Min-Chul; Cho, Min Kyoung; Lee, You Ri; Quan, Fu-Shi; Song, Jae-Min; Lee, Sujin; Moore, Martin L; Kang, Sang-Moo
Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-γ, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses. Copyright © 2015 Elsevier Inc. All rights reserved.
Ackermann, Andreas; Kugel, Daniela; Schneider, Urs; Staeheli, Peter
We previously showed that mouse adaptation of cDNA-derived Borna disease virus (BDV) strain He/80(FR) was associated exclusively with mutations in the viral polymerase complex. Interestingly, independent mouse adaptation of non-recombinant He/80 was correlated with different alterations in the polymerase and mutations in the viral glycoprotein. We used reverse genetics to demonstrate that changes in the polymerase which improve enzymatic activity represent the decisive host range mutations. The glycoprotein mutations did not confer replication competence in mice, although they slightly improved viral performance if combined with polymerase mutations. Our findings suggest that the viral polymerase restricts the host range of BDV.
Kevin A. Caillouët
Full Text Available Sensitive indicators of spatial and temporal variation in vector-host contact rates are critical to understanding the transmission and eventual prevention of arboviruses such as West Nile virus (WNV. Monitoring vector contact rates on particularly susceptible and perhaps more exposed avian nestlings may provide an advanced indication of local WNV amplification. To test this hypothesis we monitored WNV infection and vector contact rates among nestlings occupying nest boxes (primarily Eastern bluebirds; Sialia sialis, Turdidae across Henrico County, Virginia, USA, from May to August 2012. Observed host-seeking rates were temporally variable and associated with absolute vector and host abundances. Despite substantial effort to monitor WNV among nestlings and mosquitoes, we did not detect the presence of WNV in these populations. Generally low vector-nestling host contact rates combined with the negative WNV infection data suggest that monitoring transmission parameters among nestling Eastern bluebirds in Henrico County, Virginia, USA may not be a sensitive indicator of WNV activity.
Chakrabarty, Romit; Banerjee, Rituparna; Chung, Sang-Min; Farman, Mark; Citovsky, Vitaly; Hogenhout, Saskia A; Tzfira, Tzvi; Goodin, Michael
Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.
Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice▿
Cooper, David; Wright, Kevin J.; Calderon, Priscilla C.; Guo, Min; Nasar, Farooq; Johnson, J. Erik; Coleman, John W.; Lee, Margaret; Kotash, Cheryl; Yurgelonis, Irene; Natuk, Robert J.; Hendry, R. Michael; Udem, Stephen A.; Clarke, David K.
Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made. PMID:17942549
Full Text Available Immunocontraceptive vaccines may be an alternative to surgical sterilization. Dual rabies vaccination and dog population management is a helpful tool for rabies prevention. A synthetic gonadotropin-releasing hormone (GnRH peptide coupled to a carrier protein or T cell epitope is efficacious in inducing immunocontraception in a variety of mammals. However, virus-vectored GnRH recombinant vaccines have advantages over the conjugation method. In a previous in vitro study, we were able to insert a GnRH-coding sequence into the rabies virus (RABV glycoprotein (G gene, and the recombinant viruses grew to high titers in cells. Here, we further focused on the RABV G in accepting various copy numbers of GnRH. We demonstrated although RABV G protein with up to 4 copies of GnRH was well expressed, the recombinant virus was recovered only when 2 copies of GnRH (20 amino acids were incorporated into the G, indicating a possible insertion limit in making a full infectious clone. The investigation provides insight into the utility of RABV G as a carrier for small peptides and its suitability for vaccine studies. Following our previous study, we selected ERAg3p/2GnRH and tested the construct in mice. The vaccine induced ⩾80% infertility after three doses without any adjuvant, in live (8 of 10 mice infertility or inactivated (13 of 14 mice infertility formulations; while the pregnancy rate was 100% (10 of 10 mice in the controls. This initial success of immunocontraception in mice is promising, and we are now optimizing the vaccine formulation by using adjuvants and exploring novel delivery methods to minimize the dosage.
Cobleigh, Melissa A.; Bradfield, Clinton; Liu, Yuanjie; Mehta, Anand
Vesicular stomatitis virus (VSV) is a highly cytopathic virus being developed as a vaccine vector due to its ability to induce strong protective T cell and antibody responses after a single dose. However, little is known regarding the mechanisms underlying the potent immune responses elicited by VSV. We previously generated a VSV vector expressing the hepatitis B virus middle envelope surface glycoprotein (MS) that induces strong MS-specific T cell and antibody responses in mice. After synthesis in the cytoplasm, the MS protein translocates to the endoplasmic reticulum, where it forms subviral particles that are secreted from the cell. To better understand the contributions of secreted and intracellular protein to the VSV-induced immune response, we produced a vector expressing a secretion-deficient MS mutant (MSC69A) and compared the immunogenicity of this vector to that of the wild-type VSV-MS vector in mice. As expected, the MSC69A protein was not secreted from VSV-infected cells and displayed enhanced proteasome-mediated degradation. Surprisingly, despite these differences in intracellular protein processing, the T cell and antibody responses generated to MSC69A were comparable to those elicited by virus expressing wild-type MS protein. Therefore, when it is expressed from VSV, the immune responses to MS are independent of particulate antigen secretion and the turnover rate of cytoplasmic protein. These results are consistent with a model in which the immune responses to VSV are strongly influenced by the replication cycle of the vector and demonstrate that characteristics of the vector have the capacity to affect vaccine efficacy more than do the properties of the antigen itself. PMID:22345454
Bischof, Georg F; Magnani, Diogo M; Ricciardi, Michael; Shin, Young C; Domingues, Aline; Bailey, Varian K; Gonzalez-Nieto, Lucas; Rakasz, Eva G; Watkins, David I; Desrosiers, Ronald C
Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on the protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed nonstructural protein 5 (NS5), while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 × 10 5 PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited, as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two monkeys also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following high-dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against dengue virus infection. IMPORTANCE Continuing studies of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against DENV2 infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high-dose DENV2 challenge at 19 weeks
Asjes, J.; Blom-Barnhoorn, G.J.
In this study control of spread by insect vectors of non-persistent Lily symptomless virus and Lily mottle virus in lily, Tulip breaking virus in tulip, Iris mild mosaic virus, Narcissus latent virus and Iris severe mosaic virus in bulbous iris, and semi-persistent Dahlia mosaic virus and persistent
Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phosphatase vectors as helper viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo.
Zhong, Li; Chen, Linyuan; Li, Yanjun; Qing, Keyun; Weigel-Kelley, Kirsten A; Chan, Rebecca J; Yoder, Mervin C; Srivastava, Arun
Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.
Phan, Shannon I; Adam, Carolyn M; Chen, Zhenhai; Citron, Michael; Liang, Xiaoping; Espeseth, Amy S; Wang, Dai; He, Biao
Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, we developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the hemagglutinin-neuraminidase (HN) and RNA-dependent RNA polymerase (L) genes of the PIV5 genome [PIV5-RSV-F (HN-L) and PIV5-RSV-G (HN-L), respectively]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOIs) for 11 generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses. Sporadic mutations were detected in the consensus sequences of the viruses after high-MOI passages, and mutation rates increased under low-MOI-passage conditions. None of the mutations abolished antigen expression. Increased numbers of mutations correlated with increased growth rates in vitro, indicating that the viruses evolved through the course of serial passages. We also examined the in vivo stability of the vaccine candidates after a single passage in African green monkeys. No mutations were detected in the consensus sequences of viruses collected from the bronchoalveolar lavage (BAL) fluid of the animals. In vivo, mutations in RSV G and PIV5 L were found in individual isolates of PIV5-RSV-G (HN-L), but plaque isolates of PIV5-RSV-F (HN-L) had no mutations. To improve upon the PIV5-RSV-F (HN-L) candidate, additional vaccine candidates were generated in which the gene for RSV F was inserted into earlier positions in the PIV5 genome. These insertions did not negatively impact the sequence stability of the vaccine candidates. The results suggest that the RSV F and G gene insertions are stable in the PIV5 genome
Ackermann, Andreas; Guelzow, Timo; Staeheli, Peter; Schneider, Urs; Heimrich, Bernd
Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.
Steller, H; Pirrotta, V.
Drosophila larvae are rapidly killed by food containing the antibiotic G418. The bacterial gene for neomycin resistance introduced in the genome by P-mediated transformation renders larvae resistant to G418 and able to grow to fertile adults. The neo gene transcribed from the herpes thymidine kinase promoter gives low levels of resistance but high levels can be obtained using the hsp70 heat-shock promoter. We have constructed a vector for P-mediated transformation which uses this finding to a...
Wendtner, Clemens-Martin; Kurzeder, Christian; Theiss, Hans D; Kofler, David M; Baumert, Jens; Delecluse, Henri-Jacques; Janz, Annette; Hammerschmidt, Wolfgang; Hallek, Michael
Epstein-Barr virus (EBV)-based vectors have favorable features for gene transfer, including a high transduction efficiency especially for B cells, large packaging capacity up to 150 kb pairs, and ability to infect postmitotic cells. Recombinant EBV was explored for transduction of primary human B-cell chronic lymphocytic leukemia (CLL) cells. EBV vectors deleted for all oncogenic sequences and encoding terminal repeats (TR) essential for encapsidation, the lytic origin of replication (oriLyt) for DNA amplification, and the enhanced green fluorescent protein (EGFP) were packaged using an optimized, helper-virus-free method. Infectious EBV virions encoding EGFP (EBV/EGFP) with an infectious titer up to 2 x 10(6) per milliliter were generated. Primary leukemic cells from 14 patients with CLL were successfully transduced with EBV/EGFP at a very low multiplicity of infection (gp350/220. Furthermore, transduction of CLL cells with packaged EBV vectors coding for EGFP but deleted for TR sequences (TR-) did not result in EGFP expression compared to TR+ vector constructs (p = 0.009). Helper-virus-free EBV-based gene transfer vectors hold promise for development of genetic therapies for CLL patients.
Liang, Bo; Ngwuta, Joan O; Surman, Sonja; Kabatova, Barbora; Liu, Xiang; Lingemann, Matthias; Liu, Xueqiao; Yang, Lijuan; Herbert, Richard; Swerczek, Joanna; Chen, Man; Moin, Syed M; Kumar, Azad; McLellan, Jason S; Kwong, Peter D; Graham, Barney S; Collins, Peter L; Munir, Shirin
Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation.IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We
Andrade, C C; Young, K I; Johnson, W L; Villa, M E; Buraczyk, C A; Messer, W B; Hanley, K A
Each of the four serotypes of mosquito-borne dengue virus (DENV-1-4) comprises multiple, genetically distinct strains. Competitive displacement between strains within a serotype is a common feature of DENV epidemiology and can trigger outbreaks of dengue disease. We investigated the mechanisms underlying two sequential displacements by DENV-3 strains in Sri Lanka that each coincided with abrupt increases in dengue haemorrhagic fever (DHF) incidence. First, the post-DHF strain displaced the pre-DHF strain in the 1980s. We have previously shown that post-DHF is more infectious than pre-DHF for the major DENV vector, Aedes aegypti. Then, the ultra-DHF strain evolved in situ from post-DHF and displaced its ancestor in the 2000s. We predicted that ultra-DHF would be more infectious for Ae. aegypti than post-DHF but found that ultra-DHF infected a significantly lower percentage of mosquitoes than post-DHF. We therefore hypothesized that ultra-DHF had effected displacement by disseminating in Ae. aegypti more rapidly than post-DHF, but this was not borne out by a time course of mosquito infection. To elucidate the mechanisms that shape these virus-vector interactions, we tested the impact of RNA interference (RNAi), the principal mosquito defence against DENV, on replication of each of the three DENV strains. Replication of all strains was similar in mosquito cells with dysfunctional RNAi, but in cells with functional RNAi, replication of pre-DHF was significantly suppressed relative to the other two strains. Thus, differences in susceptibility to RNAi may account for the differences in mosquito infectivity between pre-DHF and post-DHF, but other mechanisms underlie the difference between post-DHF and ultra-DHF. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.
Samy,Abdallah M.; Elaagip, Arwa H.; Kenawy, Mohamed A.; Ayres, Const?ncia F. J.; Peterson, A Townsend; Soliman, Doaa E.
Rapid emergence of most vector-borne diseases (VBDs) may be associated with range expansion of vector populations. Culex quinquefasciatus Say 1823 is a potential vector of West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. We estimated the potential distribution of Cx. quinquefasciatus under both current and future climate conditions. The present potential distribution of Cx. quinquefasciatus showed high suitability across low-latitude parts of the world, reflecting th...
Vogels, Chantal B.F.; Fros, Jelke J.; Goertz, Giel; Pijlman, Gorben P.; Koenraadt, Sander
Background: Outbreaks of West Nile virus (WNV) have not occurred in northern Europe despite nearby circulation of WNV in the southern part of the continent. The main vector for WNV, the mosquito Culex (Cx.) pipiens, consists of two behaviorally distinct biotypes, pipiens and molestus, which can
Vogels, C.B.F.; Peppel, van de L.J.J.; Vliet, van A.J.H.; Westenberg, M.; Ibanez-Justicia, A.; Stroo, A.; Buijs, J.A.; Visser, T.M.; Koenraadt, C.J.M.
Culex (Cx.) pipiens mosquitoes are important vectors of West Nile virus (WNV). In Europe, the species Cx. pipiens consists of two biotypes, pipiens and molestus, which are morphologically identical, but differ in behavior. Typical behavior of the molestus biotype is the ability to remain active
Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...
Huang, Shuohao [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kawabe, Yoshinori; Ito, Akira [Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kamihira, Masamichi, E-mail: email@example.com [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)
Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.
Kushwaha, Nirbhay Kumar; Chakraborty, Supriya
Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.
Daniel T. Kinsey
Full Text Available Vector surveillance for infectious diseases is labor intensive and constantly threatened by budget decisions. We report on outcomes of an undergraduate research experience designed to build surveillance capacity for West Nile Virus (WNV in Montana (USA. Students maintained weekly trapping stations for mosquitoes and implemented assays to test for WNV in pools of Culex tarsalis. Test results were verified in a partnership with the state health laboratory and disseminated to the ArboNET Surveillance System. Combined with prior surveillance data, Cx. tarsalis accounted for 12% of mosquitoes with a mean capture rate of 74 (±SD = 118 Cx. tarsalis females per trap and a minimum infection rate of 0.3 infected mosquitoes per 1000 individuals. However, capture and infection rates varied greatly across years and locations. Infection rate, but not capture rate, was positively associated with the number of WNV human cases (Spearman’s rho = 0.94, p < 0.001. In most years, detection of the first positive mosquito pool occurred at least a week prior to the first reported human case. We suggest that undergraduate research can increase vector surveillance capacity while providing effective learning opportunities for students.
Haberman, Rebecca P; Samulski, R Jude; McCown, Thomas J
Seizure disorders present an attractive gene therapy target, particularly because viral vectors such as adeno-associated virus (AAV) and lentivirus can stably transduce neurons. When we targeted the N-methyl-D-aspartic acid (NMDA) excitatory amino acid receptor with an AAV-delivered antisense oligonucleotide, however, the promoter determined whether focal seizure sensitivity was significantly attenuated or facilitated. One potential means to circumvent this liability would be to express an inhibitory neuroactive peptide and constitutively secrete the peptide from the transduced cell. The neuropeptide galanin can modulate seizure activity in vivo, and the laminar protein fibronectin is usually secreted through a constitutive pathway. Initially, inclusion of the fibronectin secretory signal sequence (FIB) in an AAV vector caused significant gene product secretion in vitro. More importantly, the combination of this secretory signal with the coding sequence for the active galanin peptide significantly attenuated in vivo focal seizure sensitivity, even with different promoters, and prevented kainic acid-induced hilar cell death. Thus, neuroactive peptide expression and local secretion provides a new gene therapy platform for the treatment of neurological disorders.
Apparailly, F; Khoury, M; Vervoordeldonk, M J B; Adriaansen, J; Gicquel, E; Perez, N; Riviere, C; Louis-Plence, P; Noel, D; Danos, O; Douar, A-M; Tak, P P; Jorgensen, C
The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis (RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues. The aim of this study was to compare the transduction efficiency of different AAV serotypes encoding murine secreted alkaline phosphatase (mSEAP) or Escherichia coli beta-galactosidase for intraarticular gene delivery in an experimental model of arthritis. The vectors contained AAV2 terminal repeats flanking the reporter gene in an AAV1, AAV2, or AAV5 capsid, producing the pseudotypes rAAV-2/1, rAAV-2/2, and rAAV-2/5. Left knee joints of mice with collagen-induced arthritis were injected and transgene expression was analyzed by chemiluminescence or direct in situ staining of frozen sections. We show for the first time that intraarticular gene transfer with AAV- 2/5 was far more efficient than with the other serotypes tested. Transgene expression was detectable as early as 7 days after injection, reached a maximum at 21 days, and was stably expressed for at least 130 days, whereas AAV-2/1- and AAV-2/2-mediated expression levels were barely detectable. These findings provide a practical application for future local AAV-mediated gene therapy trials in RA.
Van doninck, J.; Peters, J.; De Baets, B.; Ducheyne, E.; Verhoest, N. E. C.
Bluetongue is a viral vector-borne disease transmitted between hosts, mostly cattle and small ruminants, by some species of Culicoides midges. Within the Mediterranean basin, C. imicola is the main vector of the bluetongue virus. The spatial distribution of this species is limited by a number of environmental factors, including temperature, soil properties and land cover. The identification of zones at risk of bluetongue outbreaks thus requires detailed information on these environmental factors, as well as appropriate epidemiological modelling techniques. We here give an overview of the environmental factors assumed to be constraining the spatial distribution of C. imicola, as identified in different studies. Subsequently, remote sensing products that can be used as proxies for these environmental constraints are presented. Remote sensing data are then used together with species occurrence data from the Spanish Bluetongue National Surveillance Programme to calibrate a supervised learning model, based on Random Forests, to model the probability of occurrence of the C. imicola midge. The model will then be applied for a pixel-based prediction over the Iberian peninsula using remote sensing products for habitat characterization.
Yang, Lin; Li, Juan; Xiao, Xiao
Adeno-associated virus (AAV) is emerging as a vector of choice for muscle gene therapy because of its effective and stable transduction in striated muscles. AAV naturally evolve into multiple serotypes with diverse capsid gene sequences that are apparently the determinants of their tissue tropism and infectivity. Certain AAV serotypes show robust gene transfer upon direct intramuscular injection, while others are effective in crossing the endothelial barrier to reach muscle when delivered intravenously. Muscular dystrophy gene therapy requires efficient body-wide muscle gene transfer. However, preferential liver transduction by nearly all natural AAV serotypes could be an undesirable feature for muscle-directed applications, especially by means of systemic gene delivery. Here we describe a method of in vitro evolution and in vivo selection of AAV capsids that target striated muscles and detarget the liver. Using DNA shuffling technology, we have generated a capsid gene library by in vitro scrambling and shuffling the capsid genes of natural AAV1 to AAV9. To minimize the bias and limitation of in vitro screening on culture cells, we performed direct in vivo panning in adult mice after intravenous injection of the shuffled capsid library that packaged their own coding sequences. The AAV variants enriched in the heart and muscle are retrieved by capsid gene PCR and subsequently characterized for their tissue tropisms. This directed evolution and in vivo selection method should be useful in generating novel gene therapy vectors for muscle and heart and other tissues.
Full Text Available The yellow fever (YF virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed
Hodge, Simon; Powell, Glen
Plant pathogens can induce symptoms that affect the performance of insect herbivores utilizing the same host plant. Previous studies examining the effects of infection of tic bean, Vicia faba L. (Fabales: Fabaceae), by pea enation mosaic virus (PEMV), an important disease of legume crops, indicated there were no changes in the growth and reproductive rate of its primary vector the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae). Here, we report the results of laboratory experiments investigating how A. pisum responded to PEMV infection of a different host plant, Pisum sativum L., at different stages of symptom development. Aphid growth rate was negatively related to the age of the host plant, but when they were introduced onto older plants with well-developed PEMV symptoms they exhibited a higher growth rate compared to those developing on uninfected plants of the same age. In choice tests using leaf discs A. pisum showed a strong preference for discs from PEMV-infected peas, probably in response to visual cues from the yellowed and mottled infected leaves. When adults were crowded onto leaves using clip-cages they produced more winged progeny on PEMV-infected plants. The results indicate that PEMV produces symptoms in the host plant that can enhance the performance of A. pisum as a vector, modify the production of winged progeny and affect their spatial distribution. The findings provide further evidence that some insect vector/plant pathogen interactions could be regarded as mutualistic rather than commensal when certain conditions regarding the age, stage of infection and species of host plant are met. PMID:21067425
Full Text Available Citrus Tristeza Virus (CTV disease is a silent killer, which threatens to decrease productivity, quality and even death of citrus plants and the erosion of genetic resources. Spreading in the field very quickly by the intermediate insect vector pest, aphid (Toxoptera citricida, T. Aurantii and A. Gosypii. The microbes studied for potential biopesticide candidates are: Beauveria bassiana and Hirsutella citriformis, and Metarhizium anisopliae (Metch Sorokin previously reported to control Diaphorina citri pests resulting effectiveness of > 25% and was able to suppress yield loss up to 10%. The objectives of the study examined the effectiveness of entomopathogen in controlling the pest of CTV vector, Toxoptera citricida, in the laboratory and screen house, to findout the physiological, biochemical and molecular physiology of entomopathogen. The results showed that the best entomopathogen suspension concentration was B.bassiana 106 followed by H. citriformis 106 and M. anisopliae 106. Entomopatogen B. bassiana and H. citriformis effectively controled the CTV vector pest in the laboratory. In the semi-field experiments at the screen house, the most effective result was H.citriformis 106 and the combination of H.citriformis 106 + B.bassiana 106, killing up to 50% and 100% on day 7th H.citriformis had the most physiological character, was able to develop optimally at a temperature of 20-400C and humidity between 60-80%. The biochemical character of the entomopathogenic fungus B.bassiana contained cellulase enzyme and phosphate solvent and IAA hormone, at most compared to the others. H.citriformis had not been found to contain enzymes and hormones. The molecular biochemical characterization of entomopathogenic fungi using FS1 and NS2 primers more clearly distinguished isolates and entomopathogenic species.
Skountzou, Ioanna; Koutsonanos, Dimitrios G; Kim, Jin Hyang; Powers, Ryan; Satyabhama, Lakshmipriyadarshini; Masseoud, Feda; Weldon, William C; Martin, Maria Del Pilar; Mittler, Robert S; Compans, Richard; Jacob, Joshy
The 2009 H1N1 influenza virus outbreak is the first pandemic of the twenty-first century. Epidemiological data reveal that of all the people afflicted with H1N1 virus, 60 y old have pre-existing neutralizing Abs against the 2009 H1N1 virus. This finding suggests that influenza strains that circulated 50-60 y ago might provide cross-protection against the swine-origin 2009 H1N1 influenza virus. To test this, we determined the ability of representative H1N1 influenza viruses that circulated in the human population from 1930 to 2000, to induce cross-reactivity to and cross-protection against the pandemic swine-origin H1N1 virus, A/California/04/09. We show that exposure of mice to the 1947 virus, A/FM/1/47, or the 1934 virus, A/PR/8/34, induced robust cross-protective immune responses and these mice were protected against a lethal challenge with mouse-adapted A/California/04/09 H1N1 virus. Conversely, we observed that mice exposed to the 2009 H1N1 virus were protected against a lethal challenge with mouse-adapted 1947 or 1934 H1N1 viruses. In addition, exposure to the 2009 H1N1 virus induced broad cross-reactivity against H1N1 as well as H3N2 influenza viruses. Finally, we show that vaccination with the older H1N1 viruses, particularly A/FM/1/47, confers protective immunity against the 2009 pandemic H1N1 virus. Taken together, our data provide an explanation for the decreased susceptibility of the elderly to the 2009 H1N1 outbreak and demonstrate that vaccination with the pre-1950 influenza strains can cross-protect against the pandemic swine-origin 2009 H1N1 influenza virus.
Sena-Esteves, Miguel; Saeki, Yoshinaga; Camp, Sara M.; Chiocca, E. Antonio; Breakefield, Xandra O.
We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery. PMID:10559361
Vandenberghe, Luk H; Xiao, Ru; Lock, Martin; Lin, Jianping; Korn, Michael; Wilson, James M
Vectors based on adeno-associated virus (AAV) are the subject of increasing interest as research tools and agents for in vivo gene therapy. A current limitation on the technology is the versatile and scalable manufacturing of vector. On the basis of experience with AAV2-based vectors, which remain strongly cell associated, AAV vector particles are commonly harvested from cell lysates, and must be extensively purified for use. We report here that vectors based on other AAV serotypes, including AAV1, AAV8, and AAV9, are found in abundance in, and can be harvested from, the medium of production cultures carried out with or without serum. For AAV2, this difference in compartmentalization is largely due to the affinity of the AAV2 particle for heparin, because an AAV2 variant in which the heparin-binding motif has been ablated gives higher yields and is efficiently released from cells. Vector particles isolated from the culture medium appear to be functionally equivalent to those purified from cell lysates in terms of transduction efficiency in vitro and in vivo, immunogenicity, and tissue tropism. Our findings will directly lead to methods for increasing vector yields and simplifying production processes for AAV vectors, which should facilitate laboratory-scale preparation and large-scale manufacture.
Full Text Available We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i low isoelectric point of a capsid and (ii relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.
Hastie, Eric; Samulski, R Jude
An estimated 25 million Americans are living with rare diseases. Adeno-associated virus (AAV)-mediated gene therapy is an emerging therapeutic option for the more than 7,000 identified rare diseases. This paper highlights the benefits of AAV therapy compared to conventional small molecules, discusses current pre-clinical and clinical applications of AAV-mediated gene therapy, and offers insights into cutting edge research that will shape the future of AAV for broad therapeutic use. In this review the biology of AAV and our ability to generate disease-specific variants is summarized. Limitations of current therapy are reviewed, with an emphasis on immune detection of virus, viral tropism and tissue targeting, and limitations of gene expression. Information for this review was found using PubMed and clinicaltrials.gov. Currently the scope of clinical trials of AAV gene therapy is concentrated in an array of phase I/II safety trials with less than two dozen rare diseases featured. Pre-clinical, translational studies are expanding in number as developments within the last decade have made generation of improved AAV vectors available to more researchers. Further, one bottleneck that is being overcome is the availability of disease models, which will allow for improved preclinical testing and advancement of AAV to more clinical applications.
Gabriella Dias Florencio
Full Text Available Recombinant adeno-associated viruses (rAAV are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.
Bartlett, J S; Kleinschmidt, J; Boucher, R C; Samulski, R J
We have developed a system for the targeted delivery of adeno-associated virus (AAV) vectors. Targeting is achieved via a bispecific F(ab')2 antibody that mediates a novel interaction between the AAV vector and a specific cell surface receptor expressed on human megakaryocytes. Targeted AAV vectors were able to transduce megakaryocyte cell lines, DAMI and MO7e, which were nonpermissive for normal AAV infection, 70-fold above background and at levels equivalent to permissive K562 cells. Transduction was shown to occur through the specific interaction of the AAV vector-bispecific F(ab')2 complex and cell-associated targeting receptor. Importantly, targeting appeared both selective and restrictive as the endogenous tropism of the AAV vector was significantly reduced. Binding and internalization through the alternative receptor did not alter subsequent steps (escape from endosomes, migration to nucleus, or uncoating) required to successfully transduce target cells. These results demonstrate that AAV vectors can be targeted to a specific cell population and that transduction can be achieved by circumventing the normal virus receptor.
Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking
A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic...
Agha, Sheila B; Chepkorir, Edith; Mulwa, Francis; Tigoi, Caroline; Arum, Samwel; Guarido, Milehna M; Ambala, Peris; Chelangat, Betty; Lutomiah, Joel; Tchouassi, David P; Turell, Michael J; Sang, Rosemary
In April, 2004, chikungunya virus (CHIKV) re-emerged in Kenya and eventually spread to the islands in the Indian Ocean basin, South-East Asia, and the Americas. The virus, which is often associated with high levels of viremia in humans, is mostly transmitted by the urban vector, Aedes aegypti. The expansion of CHIKV presents a public health challenge both locally and internationally. In this study, we investigated the ability of Ae. aegypti mosquitoes from three distinct cities in Kenya; Mombasa (outbreak prone), Kisumu, and Nairobi (no documented outbreak) to transmit CHIKV. Aedes aegypti mosquito populations were exposed to different doses of CHIKV (105.6-7.5 plaque-forming units[PFU]/ml) in an infectious blood meal. Transmission was ascertained by collecting and testing saliva samples from individual mosquitoes at 5, 7, 9, and 14 days post exposure. Infection and dissemination were estimated by testing body and legs, respectively, for individual mosquitoes at selected days post exposure. Tissue culture assays were used to determine the presence of infectious viral particles in the body, leg, and saliva samples. The number of days post exposure had no effect on infection, dissemination, or transmission rates, but these rates increased with an increase in exposure dose in all three populations. Although the rates were highest in Ae. aegypti from Mombasa at titers ≥106.9 PFU/ml, the differences observed were not statistically significant (χ2 ≤ 1.04, DF = 1, P ≥ 0.31). Overall, about 71% of the infected mosquitoes developed a disseminated infection, of which 21% successfully transmitted the virus into a capillary tube, giving an estimated transmission rate of about 10% for mosquitoes that ingested ≥106.9 PFU/ml of CHIKV. All three populations of Ae. aegypti were infectious as early as 5-7 days post exposure. On average, viral dissemination only occurred when body titers were ≥104 PFU/ml in all populations. Populations of Ae. aegypti from Mombasa, Nairobi
Linz, Lucas B; Liu, Sijun; Chougule, Nanasaheb P; Bonning, Bryony C
Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and surface plasmon resonance were used to confirm and characterize CP-APN interaction. PEMV virions and a peptide comprised of PEMV CP fused to a proline-rich hinge (-P-) and green fluorescent protein (CP-P-GFP) specifically bound to APN. Recombinant APN expressed in Sf9 cells resulted in internalization of CP-P-GFP, which was visualized by confocal microscopy; such internalization is an expected hallmark of a functional gut receptor. Finally, in assays with aphid gut-derived brush border membrane vesicles, binding of CP-P-GFP competed with binding of GBP3.1, a peptide previously demonstrated to bind to APN in the aphid gut and to impede PEMV uptake into the hemocoel; this finding supports the hypothesis that GBP3.1 and PEMV bind to and compete for the same APN receptor. These in vitro data combined with previously published in vivo experiments (S. Liu, S. Sivakumar, W. O. Sparks, W. A. Miller, and B. C. Bonning, Virology 401:107-116, 2010, http://dx.doi.org/10.1016/j.virol.2010.02.009) support the identification of APN as the first receptor in a plant virus vector. Knowledge of this receptor will provide for technologies based on PEMV-APN interaction designed to block plant virus transmission and to suppress aphid populations. A significant proportion of global food production is lost to insect pests. Aphids, in addition to weakening plants by feeding on their sap, are responsible for transmitting about half of the plant viruses vectored by insects. Growers rely heavily on the
Tsuda, Yoshimi; Caposio, Patrizia; Parkins, Christopher J; Botto, Sara; Messaoudi, Ilhem; Cicin-Sain, Luka; Feldmann, Heinz; Jarvis, Michael A
Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes. We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)). MCMV/ZEBOV-NP(CTL) induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection. This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.
Full Text Available Human outbreaks of Ebola virus (EBOV are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV vector expressing a CD8+ T cell epitope from the nucleoprotein (NP of Zaire ebolavirus (ZEBOV (MCMV/ZEBOV-NP(CTL. MCMV/ZEBOV-NP(CTL induced high levels of long-lasting (>8 months CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.
McKimm-Breschkin, Jennifer L; Fry, Alicia M
The International Society for Influenza and other Respiratory Virus Diseases (isirv) held its 4th Antiviral Group Conference at the University of Texas on 2-4 June, 2015. With emerging resistance to the drugs currently licensed for treatment and prophylaxis of influenza viruses, primarily the neuraminidase inhibitor oseltamivir phosphate (Tamiflu) and the M2 inhibitors amantadine and rimantadine, and the lack of effective interventions against other respiratory viruses, the 3-day programme focused on the discovery and development of inhibitors of several virus targets and key host cell factors involved in virus replication or mediating the inflammatory response. Virus targets included the influenza haemagglutinin, neuraminidase and M2 proteins, and both the respiratory syncytial virus and influenza polymerases and nucleoproteins. Therapies for rhinoviruses and MERS and SARS coronaviruses were also discussed. With the emerging development of monoclonal antibodies as therapeutics, the potential implications of antibody-dependent enhancement of disease were also addressed. Topics covered all aspects from structural and molecular biology to preclinical and clinical studies. The importance of suitable clinical trial endpoints and regulatory issues were also discussed from the perspectives of both industry and government. This meeting summary provides an overview, not only for the conference participants, but also for those interested in the current status of antivirals for respiratory viruses. Copyright © 2016 Elsevier B.V. All rights reserved.
Agha, Sheila B; Tchouassi, David P; Bastos, Armanda D S; Sang, Rosemary
The transmission patterns of dengue (DENV) and yellow fever (YFV) viruses, especially in urban settings, are influenced by Aedes (Stegomyia) mosquito abundance and behavior. Despite recurrent dengue outbreaks on the Kenyan coast, these parameters remain poorly defined in this and other areas of contrasting dengue endemicity in Kenya. In assessing the transmission risk of DENV/YFV in three Kenyan cities, we determined adult abundance and resting habits of potential Aedes (Stegomyia) vectors in Kilifi (dengue-outbreak prone), and Nairobi and Kisumu (no dengue outbreaks reported). In addition, mosquito diversity, an important consideration for changing mosquito-borne disease dynamics, was compared. Between October 2014 and June 2016, host-seeking adult mosquitoes were sampled using CO2-baited BG-Sentinel traps (12 traps daily) placed in vegetation around homesteads, across study sites in the three major cities. Also, indoor and outdoor resting mosquitoes were sampled using Prokopack aspirators. Three samplings, each of five consecutive days, were conducted during the long-rains, short-rains and dry season for each city. Inter-city and seasonal variation in mosquito abundance and diversity was evaluated using general linear models while mosquito-resting preference (indoors vs outdoors) was compared using Chi-square test. Aedes aegypti, which comprised 60% (n = 7772) of the total 12,937 host-seeking mosquitoes collected, had comparable numbers in Kisumu (45.2%, n = 3513) and Kilifi (37.7%, n = 2932), both being significantly higher than Nairobi (17.1%, n = 1327). Aedes aegypti abundance was significantly lower in the short-rains and dry season relative to the long-rains (P < 0.0001). Aedes bromeliae, which occurred in low numbers, did not differ significantly between seasons or cities. Mosquito diversity was highest during the long-rains and in Nairobi. Only 10% (n = 43) of the 450 houses aspirated were found positive for resting Ae. aegypti
Garcia-Moreno, Manuel; Sanz, Miguel Angel; Carrasco, Luis
Sindbis virus (SINV) mRNAs contain several motifs that participate in the regulation of their translation. We have discovered a motif at the 3′ untranslated region (UTR) of viral mRNAs, constituted by three repeated sequences, which is involved in the translation of both SINV genomic and subgenomic mRNAs in insect, but not in mammalian cells. These data illustrate for the first time that an element present at the 3′-UTR confers translatability to mRNAs from an animal virus in a cell-specific manner. Sequences located at the beginning of the 5′-UTR may also regulate SINV subgenomic mRNA translation in both cell lines in a context of infection. Moreover, a replicon derived from Sleeping disease virus, an alphavirus that have no known arthropod vector for transmission, is much more efficient in insect cells when the repeated sequences from SINV are inserted at its 3′-UTR, due to the enhanced translatability of its mRNAs. Thus, these findings provide a clue to understand, at the molecular level, the evolution of alphaviruses and their host range. PMID:26755446
Garcia-Moreno, Manuel; Sanz, Miguel Angel; Carrasco, Luis
Sindbis virus (SINV) mRNAs contain several motifs that participate in the regulation of their translation. We have discovered a motif at the 3' untranslated region (UTR) of viral mRNAs, constituted by three repeated sequences, which is involved in the translation of both SINV genomic and subgenomic mRNAs in insect, but not in mammalian cells. These data illustrate for the first time that an element present at the 3'-UTR confers translatability to mRNAs from an animal virus in a cell-specific manner. Sequences located at the beginning of the 5'-UTR may also regulate SINV subgenomic mRNA translation in both cell lines in a context of infection. Moreover, a replicon derived from Sleeping disease virus, an alphavirus that have no known arthropod vector for transmission, is much more efficient in insect cells when the repeated sequences from SINV are inserted at its 3'-UTR, due to the enhanced translatability of its mRNAs. Thus, these findings provide a clue to understand, at the molecular level, the evolution of alphaviruses and their host range.
Hunt Richard H
Full Text Available Abstract Background Pyrethroid resistance in Anopheles funestus populations has led to an increase in malaria transmission in southern Africa. Resistance has been attributed to elevated activities of cytochrome P450s but the molecular basis underlying this metabolic resistance is unknown. Microsatellite and SNP markers were used to construct a linkage map and to detect a quantitative trait locus (QTL associated with pyrethroid resistance in the FUMOZ-R strain of An. funestus from Mozambique. Results By genotyping 349 F2 individuals from 11 independent families, a single major QTL, rp1, at the telomeric end of chromosome 2R was identified. The rp1 QTL appears to present a major effect since it accounts for more than 60% of the variance in susceptibility to permethrin. This QTL has a strong additive genetic effect with respect to susceptibility. Candidate genes associated with pyrethroid resistance in other species were physically mapped to An. funestus polytene chromosomes. This showed that rp1 is genetically linked to a cluster of CYP6 cytochrome P450 genes located on division 9 of chromosome 2R and confirmed earlier reports that pyrethroid resistance in this strain is not associated with target site mutations (knockdown resistance. Conclusion We hypothesize that one or more of these CYP6 P450s clustered on chromosome 2R confers pyrethroid resistance in the FUMOZ-R strain of An. funestus.
Huo, Tiancheng; Qi, Li; Zhang, Buyun; Chen, Zhongping
Light carries both spin and orbital angular momentum (OAM) and the superpositions of these two dynamical properties have found many applications. Many techniques exist to create such light sources but none allow their creation at the femtosecond fiber laser. Here we report on a novel mode-locked Ytterbium-doped fiber laser that generates femtosecond pulses with generalized vector vortex states. The controlled generation of such pulses such as azimuthally and radially polarized light with definite orbital angular momentum modes are demonstrated. A unidirectional ring cavity constructed with the Yb-doped fiber placed at the end of the fiber section to reduces unnecessary nonlinear effects is employed for self-starting operation. Pairs of diffraction gratings are used for compensating the normal group velocity dispersion of the fiber and other elements. Mode-locked operation is achieved based on nonlinear polarization evolution, which is mainly implemented with the single mode fiber, the bulk wave plates and the variable spiral plates (q-plate with topological charge q=0.5). The conversion from spin angular momentum to the OAM and reverse inside the laser cavity are realized by means of a quarter-wave plate and a q-plate so that the polarization control was mapped to OAM mode control. The fiber laser is diode pumped by a wavelength-division multiplexing coupler, which leads to excellent stability and portability.
Medeiros, Ricardo B; Resende, Renato de O; de Avila, Antonio Carlos
Tospoviruses have the ability to infect plants and their insect vectors. Tomato spotted wilt virus (TSWV), the type species in the Tospovirus genus, infects its most important insect vector, Frankliniella occidentalis, the western flower thrips (WFT). However, no detrimental effects on the life cycle or cytopathological changes have been reported in the WFT after TSWV infection, and relatively few viral particles can be observed even several days after infection. We hypothesized that TSWV infection triggers an immune response in the WFT. Using subtractive cDNA libraries to probe WFT DNA macroarrays, we found that the WFT's immune system is activated by TSWV infection. The activated genes included (i) those encoding antimicrobial peptides, such as defensin and cecropin; (ii) genes involved in pathogen recognition, such as those encoding lectins; (iii) those encoding receptors that activate the innate immune response, such as Toll-3; and (iv) those encoding members of signal transduction pathways activated by Toll-like receptors, such as JNK kinase. Transcriptional upregulation of these genes after TSWV infection was confirmed by Northern analysis, and the kinetics of the immune response was measured over time. Several of the detected genes were activated at the same time that viral replication was first detected by reverse transcription-PCR. To our knowledge, this is the first report of the activation of an insect vector immune response by a plant virus. The results may lead to a better understanding of insects' immune responses against viruses and may help in the future development of novel control strategies against plant viruses, as well as human and animal viruses transmitted by insect vectors.
Marini, Giovanni; Rosá, Roberto; Pugliese, Andrea; Heesterbeek, Hans
In this study, we develop a model to investigate how ecological factors might affect the dynamics of a vector-borne pathogen in a population composed by different hosts which interact with each other. Specifically, we consider the case when different host species compete with each other, as they share the same habitat, and the vector might have different feeding preference, which can also be time dependent. As a prototypical example, we apply our model to study the invasion and spread, during a typical season, of West Nile virus in an ecosystem composed of two competent avian host species and possibly of dead-end host species. We found that competition and vector feeding preferences can profoundly influence pathogen invasion, influencing its probability to start an epidemic, and influencing transmission rates. Finally, when considering time-dependent feeding preferences, as observed in the field, we noted that the virus circulation could be amplified and that the timing of epidemic peaks could be changed. Our work highlights that ecological interactions between hosts can have a profound influence on the dynamics of the pathogen and that, when modeling vector-borne infections, vector feeding behavior should, for this reason, be carefully evaluated. Copyright © 2016 Elsevier Ltd. All rights reserved.
Rutledge, Elizabeth A.; Halbert, Christine L.; Russell, David W.
Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, a...
Full Text Available Abstract Background Chronic hepatitis B virus (HBV infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA in a mouse model for HBV replication. Methods Hepatitis B surface antigen (HBsAg and hepatitis B e antigen (HBeAg in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA technique, hepatitis B core antigen (HBcAg and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR and quantitative real-time PCR respectively. Results Co-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group. Conclusion Lentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.
Mary K Mills
Full Text Available Culicoides sonorensis biting midges are confirmed vectors of epizootic hemorrhagic disease virus (EHDV, which causes mortality in white-tailed deer and ruminant populations. Currently, of the seven EHDV serotypes, only 1, 2, and 6 are detected in the USA, and very few studies have focused on the infection time course of these serotypes within the midge. The objective of this current research was to characterize EHDV-2 infection within the midge by measuring infection prevalence, virus dissemination, and viral load over the course of infection. Midges were fed a blood meal containing 106.9 PFU/ml EHDV-2, collected every 12 h from 0-2 days post feeding (dpf and daily from 3-10 dpf, and cohorts of 20 C. sonorensis were processed using techniques that assessed EHDV infection and dissemination. Cytopathic effect assays and quantitative (qPCR were used to determine infection prevalence, revealing a 50% infection rate by 10 dpf using both methods. Using immunohistochemistry, EHDV-2 infection was detectable at 5 dpf, and shown to disseminate from the midgut to other tissues, including fat body, eyes, and salivary glands by 5 dpf. Stain intensity increased from 5-8 dpf, indicating replication of EHDV-2 in secondary infection sites after dissemination. This finding is also supported by trends in viral load over time as determined by plaque assays and qPCR. An increase in titer between 4-5 dpf correlated with viral replication in the midgut as seen with staining at day 5, while the subsequent gradual increase in viral load from 8-10 dpf suggested viral replication in midges with disseminated infection. Overall, the data presented herein suggest that EHDV-2 disseminates via the hemolymph to secondary infection sites throughout the midge and demonstrate a high potential for transmission at five days at 25°C after an infective blood-meal.
Wang, Min; Jokinen, Jenny; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S
Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c + /CD8 + dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
Full Text Available It is known that plant viruses can change the performance of their vectors. However, there have been no reports on whether or how a semipersistent plant virus manipulates the feeding behaviors of its whitefly vectors. Cucurbit chlorotic yellows virus (CCYV (genus Crinivirus, family Closteroviridae is an emergent plant virus in many Asian countries and is transmitted specifically by B and Q biotypes of tobacco whitefly, Bemisia tabaci (Gennadius, in a semipersistent manner. In the present study, we used electrical penetration graph (EPG technique to investigate the effect of CCYV on the feeding behaviors of B. tabaci. The results showed that CCYV altered feeding behaviors of both biotypes and sexes of B. tabaci with different degrees. CCYV had stronger effects on feeding behaviors of Q biotype than those of B biotype, by increasing duration of phloem salivation and sap ingestion, and could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, with more phloem ingestion in Q biotype males and more non-phloem probing in B biotype males than their respective females. With regard to feeding behaviors related to virus transmission, these results indicated that, when carrying CCYV, B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females.
Lu, Shaohua; Li, Jingjing; Wang, Xueli; Song, Danyang; Bai, Rune; Shi, Yan; Gu, Qinsheng; Kuo, Yen-Wen; Falk, Bryce W.; Yan, Fengming
It is known that plant viruses can change the performance of their vectors. However, there have been no reports on whether or how a semipersistent plant virus manipulates the feeding behaviors of its whitefly vectors. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus, family Closteroviridae) is an emergent plant virus in many Asian countries and is transmitted specifically by B and Q biotypes of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. In the present study, we used electrical penetration graph (EPG) technique to investigate the effect of CCYV on the feeding behaviors of B. tabaci. The results showed that CCYV altered feeding behaviors of both biotypes and sexes of B. tabaci with different degrees. CCYV had stronger effects on feeding behaviors of Q biotype than those of B biotype, by increasing duration of phloem salivation and sap ingestion, and could differentially manipulate feeding behaviors of males and females in both biotype whiteflies, with more phloem ingestion in Q biotype males and more non-phloem probing in B biotype males than their respective females. With regard to feeding behaviors related to virus transmission, these results indicated that, when carrying CCYV, B. tabaci Q biotype plays more roles than B biotype, and males make greater contribution than females. PMID:28098749
Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-\\/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg\\/dl) was prevented for >6 months by the dsAAV2\\/7, dsAAV2\\/8, and dsAAV2\\/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2\\/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2\\/7 and dsAAV2\\/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2\\/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-\\/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2\\/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2\\/9 vector. Hepatorenal correction in G6pase (-\\/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.
Wang, Wei; Zhao, Wan; Li, Jing; Luo, Lan; Kang, Le; Cui, Feng
No evidence has shown whether insect-borne viruses manipulate the c-Jun N-terminal kinase (JNK) signaling pathway of vector insects. Using a system comprising the plant virus Rice stripe virus (RSV) and its vector insect, the small brown planthopper, we have studied the response of the vector insect’s JNK pathway to plant virus infection. We found that RSV increased the level of Tumor Necrosis Factor-α and decreased the level of G protein Pathway Suppressor 2 (GPS2) in the insect vector. The virus capsid protein competitively bound GPS2 to release it from inhibiting the JNK activation machinery. We confirmed that JNK activation promoted RSV replication in the vector, whereas JNK inhibition caused a significant reduction in virus production and thus delayed the disease incidence of plants. These findings suggest that inhibition of insect vector JNK may be a useful strategy for controling the transmission of plant viruses. DOI: http://dx.doi.org/10.7554/eLife.26591.001 PMID:28716183
Wiegand, Marian Alexander; Gori-Savellini, Gianni; Gandolfo, Claudia; Papa, Guido; Kaufmann, Christine; Felder, Eva; Ginori, Alessandro; Disanto, Maria Giulia; Spina, Donatella; Cusi, Maria Grazia
Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated.IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity
Duraffour, Sophie; Andrei, Graciela; Topalis, Dimitri; Krečmerová, Marcela; Crance, Jean-Marc; Garin, Daniel; Snoeck, Robert
Cidofovir or (S)-HPMPC is one of the three antiviral drugs that might be used for the treatment of orthopoxvirus infections. (S)-HPMPC and its 2,6-diaminopurine counterpart, (S)-HPMPDAP, have been described to select, in vitro, for drug resistance mutations in the viral DNA polymerase (E9L) gene of vaccinia virus (VACV). Here, to extend our knowledge of drug resistance development among orthopoxviruses, we selected, in vitro, camelpox viruses (CMLV) resistant to (S)-HPMPDAP and identified a single amino acid change, T831I, and a double mutation, A314V+A684V, within E9L. The production of recombinant CMLV and VACV carrying these amino acid substitutions (T831I, A314V, or A314V+A684V) demonstrated clearly their involvement in conferring reduced sensitivity to viral DNA polymerase inhibitors, including (S)-HPMPDAP. Both CMLV and VACV harboring the A314V change showed comparable drug-susceptibility profiles to various antivirals and similar impairments in viral growth. In contrast, the single change T831I and the double change A314V+A684V in VACV were responsible for increased levels of drug resistance and for cross-resistance to viral DNA polymerase antivirals that were not observed with their CMLV counterparts. Each amino acid change accounted for an attenuated phenotype of VACV in vivo. Modeling of E9L suggested that the T→I change at position 831 might abolish hydrogen bonds between E9L and the DNA backbone and have a direct impact on the incorporation of the acyclic nucleoside phosphonates. Our findings demonstrate that drug-resistance development in two related orthopoxvirus species may impact drug-susceptibility profiles and viral fitness differently.
Liu, Yuhe; Okada, Takashi; Sheykholeslami, Kianoush; Shimazaki, Kuniko; Nomoto, Tatsuya; Muramatsu, Shin-Ichi; Kanazawa, Takeharu; Takeuchi, Koichi; Ajalli, Rahim; Mizukami, Hiroaki; Kume, Akihiro; Ichimura, Keiichi; Ozawa, Keiya
Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken beta-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.
Chen, Yong; Ott, Christopher J; Townsend, Kay; Subbaiah, Papasani; Aiyar, Ashok; Miller, William M
Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl beta cyclodextrin) on the production of Molony murine leukemia virus retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than two-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.
Tian, Ji; Pei, Haixia; Zhang, Shuai; Chen, Jiwei; Chen, Wen; Yang, Ruoyun; Meng, Yonglu; You, Jie; Gao, Junping; Ma, Nan
Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3' terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV-GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV-GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV-GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75-80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants.
Tian, Ji; Pei, Haixia; Ma, Nan
Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3’ terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV–GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV–GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV–GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75–80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants. PMID:24218330
Michal Segoli; Hoffmann, Ary A.; Jane Lloyd; Omodei, Gavin J.; Scott A. Ritchie
Background The bacterial endosymbiont Wolbachia blocks the transmission of dengue virus by its vector mosquito Aedes aegypti, and is currently being evaluated for control of dengue outbreaks. Wolbachia induces cytoplasmic incompatibility (CI) that results in the developmental failure of offspring in the cross between Wolbachia-infected males and uninfected females. This increases the relative success of infected females in the population, thereby enhancing the spread of the beneficial bacteri...
Lin, Shih-Chang; Liu, Wen-Chun; Lin, Yu-Fen; Huang, Yu-Hsuan; Liu, Jin-Hwang; Wu, Suh-Chin
Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to trigger severe diseases in poultry and humans, prompting efforts to develop an effective vaccine. Toward that goal, we constructed a recombinant adenovirus vector encoding influenza hemagglutin (rAd-HA) and a flagellin-containing virus-like particle (FliC-VLP). Using a murine model, we investigated a heterologous prime-boost vaccination regimen combining these two vectors. Our results indicate that priming with the rAd-HA vector followed by a FliC-VLP booster induced the highest HA-specific total IgG, IgG1and IgG2a. Maximum neutralizing antibody titers against homologous and heterologous clades of H5N1 virus strains and hemagglutination inhibition resulted from the heterologous vaccination strategy. Our results are likely to contribute to the development of more effective H5N1 vaccines. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Guo, Xiao-Xia; Zhu, Xiao-Juan; Li, Chun-Xiao; Dong, Yan-De; Zhang, Ying-Mei; Xing, Dan; Xue, Rui-De; Qin, Cheng-Feng; Zhao, Tong-Yan
The vector competence of Aedes albopictus and Aedes aegypti with regard to DEN2-43 and New Guinea C (NGC) virus strains of Dengue 2 viruses was assessed and compared. The infection and dissemination rate and distribution of DEN2-43 antigens in orally infected Ae. albopictus was investigated using the reverse transcription polymerase chain reaction and an indirect immunofluorescence assay. To better understand the initial infection, dissemination and transmission of these viral strains in vector mosquitoes, Ae. albopoictus and Ae. aegypti were fed an artificial blood meal containing either the DEN2-43 or NGC strain. There was no significant difference in the infection and dissemination rates of DEN2-43 and NGC virus strains in Ae. albopictus, however, Ae. aegypti was more susceptible to infection by NGC than DEN2-43 vrius strain. Ae. albopictus mosquitoes infected with the NGC strain developed a higher percentage of midgut infections than those infected with the DEN2-43 strain (t=2.893, df=7, P=0.024). Approximately 26.7% of midgut samples were positive for the NGC antigen 5 days after infection, and 80% of mosquitoes had infected midgets after 15 days. The NGC antigen first became evident in mosquito salivary glands on Day 5, and 40% of mosquitoes had infected salivary by Day 9. In contrast, the DEN2-43 antigen first became evident in salivary glands on Day 7. The infection rate of NGC and DEN2-43 virus strains in salivary glands were similar. These results indicate that Ae. albopictus and Ae. aegypti are moderately competent vectors for the DEN2-43 virus, which could provide basic data for the epidemiology study of dengue fever in China. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
Verheust, Céline; Goossens, Martine; Pauwels, Katia; Breyer, Didier
The modified vaccinia virus Ankara (MVA) strain is a highly attenuated strain of vaccinia virus that has been demonstrated to be safe for humans. MVA is widely considered as the vaccinia virus strain of choice for clinical investigation because of its high safety profile. It also represents an excellent candidate for use as vector system in recombinant vaccine development for gene delivery or vaccination against infectious diseases or tumours, even in immunocompromised individuals. The use of MVA and recombinant MVA vectors must comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The purpose of the present paper is to highlight some biological characteristics of MVA and MVA-based recombinant vectors and to discuss these from a biosafety point of view in the context of the European regulatory framework for genetically modified organisms with emphasis on the assessment of potential risks associated with environmental release. Copyright Â© 2012 Elsevier Ltd. All rights reserved.
Casimiro, Danilo R.; Chen, Ling; Fu, Tong-Ming; Evans, Robert K.; Caulfield, Michael J.; Davies, Mary-Ellen; Tang, Aimin; Chen, Minchun; Huang, Lingyi; Harris, Virginia; Freed, Daniel C.; Wilson, Keith A.; Dubey, Sheri; Zhu, De-Min; Nawrocki, Denise; Mach, Henryk; Troutman, Robert; Isopi, Lynne; Williams, Donna; Hurni, William; Xu, Zheng; Smith, Jeffrey G.; Wang, Su; Liu, Xu; Guan, Liming; Long, Romnie; Trigona, Wendy; Heidecker, Gwendolyn J.; Perry, Helen C.; Persaud, Natasha; Toner, Timothy J.; Su, Qin; Liang, Xiaoping; Youil, Rima; Chastain, Michael; Bett, Andrew J.; Volkin, David B.; Emini, Emilio A.; Shiver, John W.
Cellular immune responses, particularly those associated with CD3+ CD8+ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4+ and CD8+ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting. PMID:12743287
Daito, Takuji; Fujino, Kan; Honda, Tomoyuki; Matsumoto, Yusuke; Watanabe, Yohei; Tomonaga, Keizo
Borna disease virus (BDV), a nonsegmented, negative-strand RNA virus, infects a wide variety of mammalian species and readily establishes a long-lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5' untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/M-GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, ΔGLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV ΔGLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV ΔGLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions.
Daito, Takuji; Fujino, Kan; Honda, Tomoyuki; Matsumoto, Yusuke; Watanabe, Yohei; Tomonaga, Keizo
Borna disease virus (BDV), a nonsegmented, negative-strand RNA virus, infects a wide variety of mammalian species and readily establishes a long-lasting, persistent infection in brain cells. Therefore, this virus could be a promising candidate as a novel RNA virus vector enabling stable gene expression in the central nervous system (CNS). Previous studies demonstrated that the 5′ untranslated region of the genome is the only site for insertion and expression of a foreign gene. In this study, we established a novel BDV vector in which an additional transcription cassette has been inserted into an intercistronic noncoding region between the viral phosphoprotein (P) and matrix (M) genes. The recombinant BDV (rBDV) carrying green fluorescent protein (GFP) between the P and M genes, rBDV P/M-GFP, expressed GFP efficiently in cultured cells and rodent brains for a long period of time without attenuation. Furthermore, we generated a nonpropagating rBDV, ΔGLLP/M, which lacks the envelope glycoprotein (G) and a splicing intron within the polymerase gene (L), by the transcomplementation system with either transient or stable expression of the G gene. Interestingly, rBDV ΔGLLP/M established a persistent infection in cultured cells with stable expression of GFP in the absence of the expression of G. Using persistently infected rBDV ΔGLLP/M-infected cells, we determined the amino acid region in the cytoplasmic tail (CT) of BDV G important for the release of infectious rBDV particles and also demonstrated that the CT region may be critical for the generation of pseudotyped rBDV having vesicular stomatitis virus G protein. Our results revealed that the newly established BDV vector constitutes an alternative tool not only for stable expression of foreign genes in the CNS but also for understanding the mechanism of the release of enveloped virions. PMID:21937656
Full Text Available This review examines recent studies of the migration of three rice planthoppers, Laodelphax striatellus, Sogatella furcifera, and Nilaparvata lugens, in East Asia. Laodelphax striatellus has recently broken out in Jiangsu province, eastern China. The population density in the province started to increase in the early 2000s and peaked in 2004. In 2005, Rice stripe virus (RSV viruliferous rate of L. striatellus peaked at 31.3%. Since then, rice stripe disease spread severely across the whole province. Due to the migration of the RSV vectors, the rice stripe disease spread to neighboring countries Japan and Korea. An overseas migration of L. striatellus that occurred in 2008 was analyzed, when a slow-moving cold vortex, a type of low pressure system, reached western Japan from Jiangsu, carrying the insects into Japan. Subsequently the rice stripe diseases struck these areas in Japan severely. In Korea, similar situations occurred in 2009, 2011, and 2012. Their migration sources were also estimated to be in Jiangsu by backward trajectory analysis. Rice black-streaked dwarf virus, whose vector is L. striatellus, has recently re-emerged in eastern China, and the evidence for overseas migrations of the virus, just like the RSV’s migrations, has been given. A method of predicting the overseas migration of L. striatellus has been developed by Japanese, Chinese, and Korean institutes. An evaluation of the prediction showed that this method properly predicted migration events that occurred in East Asia from 2008 to 2011. Southern rice black-streaked dwarf virus (SRBSDV was first found in Guangdong province. Its vector is S. furcifera. An outbreak of SRBSDV occurred in southern China in 2009 and spread to Vietnam the same year. This disease and virus were also found in Japan in 2010. The epidemic triggered many migration studies to investigate concrete spring-summer migration routes in China, and the addition of migration sources for early arrivals in
Shao, Guohong; Greathouse, Kristin; Huang, Qin; Wang, Chiou-Miin; Sferra, Thomas J
The transfer of exogenous genetic material to cells within the gastrointestinal (GI) tract has many potential therapeutic applications. An attractive feature of the GI tract for gene transfer is its accessibility through the orogastric route. In this study, we evaluated the stability of recombinant adeno-associated virus type 2 (rAAV2) vectors within the GI tract and whether rAAV2-mediated gene transfer could be increased through manipulation of the intraluminal environment. The stability of rAAV2 vectors carrying beta-galactosidase and enhanced green fluorescence protein transgenes was determined in the presence of hydrochloric acid, pepsin, trypsin, chymotrypsin gastric fluid and intestinal fluid and after in vivo administration. For in vivo experiments, the rAAV2 vector carrying the beta-galactosidase transgene was administered perorally to FVB/NJ mice. Groups of mice received the vector alone or in combination with sodium bicarbonate and aprotinin. Gene transfer to the stomach and small intestine was evaluated by polymerase chain reaction and histochemical assays. The stability of rAAV2 was reduced by hydrochloric acid, trypsin, chymotrypsin, gastric fluid and intestinal fluid. The vector was not stable within the lumen of the GI tract. Gastric acid neutralization with sodium bicarbonate and protease inhibition with aprotinin increased the in vivo stability of the vector and the level of gene transfer to the stomach and all regions of the small bowel. In both groups of mice (vector alone and vector plus sodium bicarbonate and aprotinin), transgene-derived protein expression (beta-galactosidase) was below the level of detection of the histochemical assay. Recombinant AAV2 are adversely affected by physiological conditions within the proximal GI tract. Gastric acid neutralization and inhibition of intestinal protease activity improved rAAV2 stability and increased the level of gene transfer within the GI tract. Despite these changes, transduction of the GI tract
Hamer, Gabriel L; Chaves, Luis F; Anderson, Tavis K; Kitron, Uriel D; Brawn, Jeffrey D; Ruiz, Marilyn O; Loss, Scott R; Walker, Edward D; Goldberg, Tony L
The influence of host diversity on multi-host pathogen transmission and persistence can be confounded by the large number of species and biological interactions that can characterize many transmission systems. For vector-borne pathogens, the composition of host communities has been hypothesized to affect transmission; however, the specific characteristics of host communities that affect transmission remain largely unknown. We tested the hypothesis that vector host use and force of infection (i.e., the summed number of infectious mosquitoes resulting from feeding upon each vertebrate host within a community of hosts), and not simply host diversity or richness, determine local infection rates of West Nile virus (WNV) in mosquito vectors. In suburban Chicago, Illinois, USA, we estimated community force of infection for West Nile virus using data on Culex pipiens mosquito host selection and WNV vertebrate reservoir competence for each host species in multiple residential and semi-natural study sites. We found host community force of infection interacted with avian diversity to influence WNV infection in Culex mosquitoes across the study area. Two avian species, the American robin (Turdus migratorius) and the house sparrow (Passer domesticus), produced 95.8% of the infectious Cx. pipiens mosquitoes and showed a significant positive association with WNV infection in Culex spp. mosquitoes. Therefore, indices of community structure, such as species diversity or richness, may not be reliable indicators of transmission risk at fine spatial scales in vector-borne disease systems. Rather, robust assessment of local transmission risk should incorporate heterogeneity in vector host feeding and variation in vertebrate reservoir competence at the spatial scale of vector-host interaction.
Gabriel L Hamer
Full Text Available The influence of host diversity on multi-host pathogen transmission and persistence can be confounded by the large number of species and biological interactions that can characterize many transmission systems. For vector-borne pathogens, the composition of host communities has been hypothesized to affect transmission; however, the specific characteristics of host communities that affect transmission remain largely unknown. We tested the hypothesis that vector host use and force of infection (i.e., the summed number of infectious mosquitoes resulting from feeding upon each vertebrate host within a community of hosts, and not simply host diversity or richness, determine local infection rates of West Nile virus (WNV in mosquito vectors. In suburban Chicago, Illinois, USA, we estimated community force of infection for West Nile virus using data on Culex pipiens mosquito host selection and WNV vertebrate reservoir competence for each host species in multiple residential and semi-natural study sites. We found host community force of infection interacted with avian diversity to influence WNV infection in Culex mosquitoes across the study area. Two avian species, the American robin (Turdus migratorius and the house sparrow (Passer domesticus, produced 95.8% of the infectious Cx. pipiens mosquitoes and showed a significant positive association with WNV infection in Culex spp. mosquitoes. Therefore, indices of community structure, such as species diversity or richness, may not be reliable indicators of transmission risk at fine spatial scales in vector-borne disease systems. Rather, robust assessment of local transmission risk should incorporate heterogeneity in vector host feeding and variation in vertebrate reservoir competence at the spatial scale of vector-host interaction.
Li, Junmin; Andika, Ida Bagus; Shen, Jiangfeng; Lv, Yuanda; Ji, Yongqiang; Sun, Liying; Chen, Jianping
Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.
Nieto, Karen; Kern, Andrea; Leuchs, Barbara; Gissmann, Lutz; Müller, Martin; Kleinschmidt, Jürgen A
Cervical cancer is the second most frequent cancer among woman worldwide and is considered to be caused by infection with high-risk papilloma viruses. Genetic immunization using recombinant adeno-associated virus (rAAV) vectors has shown great promise for vaccination against human papillomavirus (HPV) infections. rAAV5, -8 and -9 vectors expressing an HPV16 L1/E7 fusion gene were generated and applied intranasally for combined prophylactic and therapeutic vaccination of mice. The rAAV5 and the rAAV9 vectors showed efficient induction of both humoral and cellular immune responses, whereas rAAV8 failed to immunize mice by the intranasal route. The L1-specific immune response evoked by expression of the L1/E7 fusion gene, however, was lower than that evoked by expression of the L1 antigen alone. This deficiency could be compensated by application of Escherichia coli heat-labile enterotoxin or monophsphoryl lipid as adjuvant upon vaccination with rAAV5-L1/E7. Coimmunization of rAAV9-L1/E7 with rAAV5-L1 or boosting of rAAV9-L1/E7 with rAAV5-L1 strongly increased L1-specific neutralizing antibody titres to levels above those achieved by vaccination with vectors expressing L1 alone. Both vectors elicited a vibrant cytotoxic T-lymphocyte response against L1 or E7. Nasal immunization with rAAV5 or rAAV9 was superior to vaccination with HPV16-L1 virus-like particles (VLPs) or HPV16-L1/E7 CVLPs with respect to humoral and cellular immune responses. Vaccination with the rAAV vectors led to a significant protection of animals against a challenge with different HPV tumour cell lines. Our results show that rAAV5 and rAAV9 vectors are promising candidates for a non-invasive nasal vaccination strategy.
Mardanova, Eugenia S; Blokhina, Elena A; Tsybalova, Liudmila M; Peyret, Hadrien; Lomonossoff, George P; Ravin, Nikolai V
Agroinfiltration of plant leaves with binary vectors carrying a gene of interest within a plant viral vector is a rapid and efficient method for protein production in plants. Previously, we constructed a self-replicating vector, pA7248AMV, based on the genetic elements of potato virus X (PVX), and have shown that this vector can be used for the expression of recombinant proteins in Nicotiana benthamiana. However, this vector is almost 18 kb long and therefore not convenient for genetic manipulation. Furthermore, for efficient expression of the target protein it should be co-agroinfiltrated with an additional binary vector expressing a suppressor of post-transcriptional gene silencing. Here, we improved this expression system by creating the novel pEff vector. Its backbone is about 5 kb shorter than the original vector and it contains an expression cassette for the silencing suppressor, P24, from grapevine leafroll-associated virus-2 alongside PVX genetic elements, thus eliminating the need of co-agroinfiltration. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein. The novel vector was used for expression of the influenza vaccine candidate, M2eHBc, consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen. Using the pEff system, M2eHBc was expressed to 5-10% of total soluble protein, several times higher than with original pA7248AMV vector. Plant-produced M2eHBc formed virus-like particles in vivo, as required for its use as a vaccine. The new self-replicating pEff vector could be used for fast and efficient production of various recombinant proteins in plants.
Jian, Y.; Silvestri, S.; Marani, M.; Saltarin, A.; Chillemi, G.
We applied a hierarchical state space model to predict the abundance of Cx.pipiens (a West Nile Virus vector) in the Po River Delta Region, Northeastern Italy. The study area has large mosquito abundance, due to a favorable environment and climate as well as dense human population. Mosquito data were collected on a weekly basis at more than 20 sites from May to September in 2010 and 2011. Cx.pipiens was the dominant species in our samples, accounting for about 90% of the more than 300,000 total captures. The hydrological component of the model accounted for evapotranspiration, infiltration and deep percolation to infer, in a 0D context, the local dynamics of soil moisture as a direct exogenous forcing of mosquito dynamics. The population model had a Gompertz structure, which included exogenous meteorological forcings and delayed internal dynamics. The models were coupled within a hierarchical statistical structure to overcome the relatively short length of the samples by exploiting the large number of concurrent observations available. The results indicated that Cx.pipiens abundance had significant density dependence at 1 week lag, which approximately matched its development time from larvae to adult. Among the exogenous controls, temperature, daylight hours, and soil moisture explained most of the dynamics. Longer daylight hours and lower soil moisture values resulted in higher abundance. The negative correlation of soil moisture and mosquito population can be explained with the abundance of water in the region (e.g. due to irrigation) and the preference for eutrophic habitats by Cx.pipien. Variations among sites were explained by land use factors as represented by distance to the nearest rice field and NDVI values: the carrying capacity decreased with increased distance to the nearest rice filed, while the maximum growth rate was positively related with NDVI. The model shows a satisfactory performance in predicting (potentially one week in advance) mosquito
Full Text Available Compared to other integrating viral vectors, foamy virus (FV vectors have distinct advantages as a gene transfer tool, including their nonpathogenicity, the ability to carry larger transgene cassettes, and increased stability of virus particles due to DNA genome formation within the virions. Proof of principle of its therapeutic utility was provided with the correction of canine leukocyte adhesion deficiency using autologous CD34+ cells transduced with FV vector carrying the canine CD18 gene, demonstrating its long-term safety and efficacy. However, infectious titers of FV-human(hCD18 were low and not suitable for manufacturing of clinical-grade product. Herein, we developed a scalable production and purification process that resulted in 60-fold higher FV-hCD18 titers from ∼1.7 × 104 to 1.0 × 106 infectious units (IU/ml. Process development improvements included use of polyethylenimine-based transfection, use of a codon-optimized gag, heparin affinity chromatography, tangential flow filtration, and ultracentrifugation, which reproducibly resulted in 5,000-fold concentrated and purified virus, an overall yield of 19 ± 3%, and final titers of 1–2 × 109 IU/ml. Highly concentrated vector allowed reduction of final dimethyl sulfoxide (DMSO concentration, thereby avoiding DMSO-induced toxicity to CD34+ cells while maintaining high transduction efficiencies. This process development results in clinically relevant, high titer FV which can be scaled up for clinical grade production.
Jerusalinsky, Diana; Baez, María Verónica; Epstein, Alberto Luis
Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures
Tian, Debin; Ni, Yan-Yan; Zhou, Lei; Opriessnig, Tanja; Cao, Dianjun; Piñeyro, Pablo; Yugo, Danielle M; Overend, Christopher; Cao, Qian; Lynn Heffron, C; Halbur, Patrick G; Pearce, Douglas S; Calvert, Jay G; Meng, Xiang-Jin
The extensive genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains is a major obstacle for vaccine development. We previously demonstrated that chimeric PRRSVs in which a single envelope gene (ORF3, ORF4, ORF5 or ORF6) was shuffled via DNA shuffling had an improved heterologous cross-neutralizing ability. In this study, we incorporate all of the individually-shuffled envelope genes together in different combinations into an infectious clone backbone of PRRSV MLV Fostera(®) PRRS. Five viable progeny chimeric viruses were rescued, and their growth characteristics were characterized in vitro. In a pilot pig study, two chimeric viruses (FV-SPDS-VR2,FV-SPDS-VR5) were found to induce cross-neutralizing antibodies against heterologous strains. A subsequent vaccination/challenge study in 72 pigs revealed that chimeric virus FV-SPDS-VR2 and parental virus conferred partial cross-protection when challenged with heterologous strains NADC20 or MN184B. The results have important implications for future development of an effective PRRSV vaccine that confers heterologous protection. Copyright © 2015 Elsevier Inc. All rights reserved.
Watson, Zachary L; Ertel, Monica K; Lewin, Alfred S; Tuli, Sonal S; Schultz, Gregory S; Neumann, Donna M; Bloom, David C
Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose
Chinnaraja, C; Viswanathan, R
.... Studies were conducted on the virus transmission by sugarcane aphid Melanaphis sacchari in sugarcane by inoculating virus-free meristem derived from micro- propagated plants of sugarcane cv Co 86032...
Meador, Lydia R; Kessans, Sarah A; Kilbourne, Jacquelyn; Kibler, Karen V; Pantaleo, Giuseppe; Roderiguez, Mariano Esteban; Blattman, Joseph N; Jacobs, Bertram L; Mor, Tsafrir S
Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Chinnaraja, C.; Viswanathan, R.
Yellow leaf caused by Sugarcane yellow leaf virus (SCYLV) is a serious constraint to sugarcane production in India and currently the disease epidemics occur on many of the susceptible varieties under field conditions. Studies were conducted on the virus transmission by sugarcane aphid Melanaphis sacchari in sugarcane by inoculating virus-free meristem derived from micro- propagated plants of sugarcane cv Co 86032 with viruliferous aphids. Virus transmission was confirmed through RT-PCR assays...
da Moura, Aires Januário Fernandes; de Melo Santos, Maria Alice Varjal; Oliveira, Claudia Maria Fontes; Guedes, Duschinka Ribeiro Duarte; de Carvalho-Leandro, Danilo; da Cruz Brito, Maria Lidia; Rocha, Hélio Daniel Ribeiro; Gómez, Lara Ferrero; Ayres, Constância Flávia Junqueira
Dengue is an arboviral disease caused by dengue virus (DENV), whose main vectors are the mosquitoes Aedes aegypti and Aedes albopictus. A. aegypti is the only DENV vector in Cape Verde, an African country that suffered its first outbreak of dengue in 2009. However, little is known about the variation in the level of vector competence of this mosquito population to the different DENV serotypes. This study aimed to evaluate the vector competence of A. aegypti from the island of Santiago, Cape Verde, to four DENV serotypes and to detect DENV vertical transmission. Mosquitoes were fed on blood containing DENV serotypes and were dissected at 7, 14 and 21 days post-infection (dpi) to detect the virus in the midgut, head and salivary glands (SG) using RT-PCR. Additionally, the number of copies of viral RNA present in the SG was determined by qRT-PCR. Furthermore, eggs were collected in the field and adult mosquitoes obtained were analyzed by RT-PCR and the platelia dengue NS1 antigen kit to detect transovarial transmission. High rates of SG infection were observed for DENV-2 and DENV-3 whereas for DENV-1, viral RNA was only detected in the midgut and head. DENV-4 did not spread to the head or SG, maintaining the infection only in the midgut. The number of viral RNA copies in the SG did not vary significantly between DENV-2 and DENV-3 or among the different periods of incubation and the various titers of DENV tested. With respect to DENV surveillance in mosquitoes obtained from the eggs collected in the field, no samples were positive. Although no DENV positive samples were collected from the field in 2014, it is important to highlight that the A. aegypti population from Santiago Islands exhibited different degrees of susceptibility to DENV serotypes. This population showed a high vector competence for DENV-2 and DENV-3 strains and a low susceptibility to DENV-1 and DENV-4. Viral RNA copies in the SG remained constant for at least 21 dpi, which may enhance the vector
Nathanson, J L; Yanagawa, Y; Obata, K; Callaway, E M
Despite increasingly widespread use of recombinant adeno-associated virus (AAV) and lentiviral (LV) vectors for transduction of neurons in a wide range of brain structures and species, the diversity of cell types within a given brain structure is rarely considered. For example, the ability of a vector to transduce neurons within a brain structure is often assumed to indicate that all neuron types within the structure are transduced. We have characterized the transduction of mouse somatosensory cortical neuron types by recombinant AAV pseudotyped with serotype 1 capsid (rAAV2/1) and by recombinant lentivirus pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein. Both vectors used human synapsin (hSyn) promoter driving DsRed-Express. We demonstrate that high titer rAAV2/1-hSyn efficiently transduces both cortical excitatory and inhibitory neuronal populations, but use of lower titers exposes a strong preference for transduction of cortical inhibitory neurons and layer 5 pyramidal neurons. In contrast, we find that VSV-G-LV-hSyn principally labels excitatory cortical neurons at the highest viral titer generated. These findings demonstrate that endogenous tropism of rAAV2/1 and VSV-G-LV can be used to obtain preferential gene expression in mouse somatosensory cortical inhibitory and excitatory neuron populations, respectively.
Guillermo León M
Full Text Available The Citrus leprosis virus CiLV-C is a quarantine disease of economic importance. Over the past 15 years, this disease has spread to several countries of Central and South America. Colombia has about 45,000 hectares of citrus planted with an annual production of 750,000 tonnes. The CiLV-C has only been detected in the departments of Meta, Casanare and recently Tolima. Meta has 4,300 hectares representing 10% of the national cultivated area, and Casanare, where CiLV-C appeared in 2004, has no more than 500 ha planted with citrus. The presence of the Citrus leprosis virus in Colombia could affect the international market for citrus, other crops and ornamental plants with the United States and other countries without the disease. The false spider mite Brevipalpus phoenicis (Geijskes (Acari: Tenuipalpidae is the main vector of the CiLV-C. Disease management is based on control programs of the vector and diminishing host plants. Chemical mite control is expensive, wasteful and generates resistance to different acaricides. This paper provides basic information on CiLV-C and its vector, advances in diagnosis and methods to control the disease and prevention of its spread
ZHU, Cheng-chu; CHEN, Shi-lin; LIU, Yu-qing; TANG, Li-jiang; BAO, Wei-guang
To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1 (ANG-1) gene and to express the ANG-1 in targeting cells. ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1, the virus stocks in high titer were harvested. The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously. Titration of rAAVANG-1 stock was 9 X 10(11)v.g/ml. The expression of ANG-1 gene was detected in transfected cells. Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells, 55% cells expressed GFP. The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells.
Schneweis, Derek J; Whitfield, Anna E; Rotenberg, Dorith
Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a circulative-propagative manner. Little is known about thrips vector response to TSWV during the infection process from larval acquisition to adult inoculation of plants. Whole-body transcriptome response to virus infection was determined for first-instar larval, pre-pupal and adult thrips using RNA-Seq. TSWV responsive genes were identified using preliminary sequence of a draft genome of F. occidentalis as a reference and three developmental-stage transcriptomes were assembled. Processes and functions associated with host defense, insect cuticle structure and development, metabolism and transport were perturbed by TSWV infection as inferred by ontologies of responsive genes. The repertoire of genes responsive to TSWV varied between developmental stages, possibly reflecting the link between thrips development and the virus dissemination route in the vector. This study provides the foundation for exploration of tissue-specific expression in response to TSWV and functional analysis of thrips gene function. Copyright Â© 2016 Elsevier Inc. All rights reserved.
Sen, Dwaipayan; Balakrishnan, Balaji; Gabriel, Nishanth; Agrawal, Prachi; Roshini, Vaani; Samuel, Rekha; Srivastava, Alok; Jayandharan, Giridhara R.
Despite significant advancements with recombinant AAV2 or AAV8 vectors for liver directed gene therapy in humans, it is well-recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer. To overcome these limitations, alternate AAV serotypes (1–10) are being rigorously evaluated. AAV5 is the most divergent (55% similarity vs. other serotypes) and like AAV1 vector is known to transduce liver efficiently. AAV1 and AAV5 vectors are also immunologically distinct by virtue of their low seroprevalence and minimal cross reactivity against pre-existing AAV2 neutralizing antibodies. Here, we demonstrate that targeted bio-engineering of these vectors, augment their gene expression in murine hepatocytes in vivo (up to 16-fold). These studies demonstrate the feasibility of the use of these novel AAV1 and AAV5 vectors for potential gene therapy of diseases like hemophilia. PMID:23665951
Full Text Available Field visits and surveys were carried out in Niger, Kano, Bauchi and Gombe states of northern Nigeria at tillering and panicle initiation stages of rice in the years 2000 and 2001 to determine the distribution, host plants and occurrence of insect vectors of Rice Yellow Mottle Virus (RYMV. Farmers' cultural practices and field situations were also assessed. Visual inspection based on the Standard Evaluation Scale (SES and enzyme linked immunosorbent assay (ELISA methods were used in detecting RYMV infection. RYMV presence was established in all the four states surveyed. The virus was widely distributed in Kano state. The insect vectors of RYMV, such as Trichispa sericea Guerin, Chaetocnema pulla Chapius, Chnootriba similis Thunberg and Conocephalus longipennis de Haan, were found in the 4 states. Outbreaks of T. sericea occurred in many farmers' fields in Kano state. RYMV was detected more frequently on Oryza sativa L. than on O. longistaminata Chev. & Roehr and Echinochloa pyramidalis Hitche and Chase.Virus infection was not established in any other grass species, sedges and broadleaf plants tested. It is evident therefore, that RYMV has a narrow host range and is found more frequently in the Oryzeae.
Kim, Min-Chul; Lee, Jong-Seok; Kwon, Young-Man; O, Eunju; Lee, Youn-Jeong; Choi, Jun-Gu; Wang, Bao-Zhong; Compans, Richard W; Kang, Sang-Moo
The influenza M2 ectodomain (M2e) is poorly immunogenic and has some amino acid changes among isolates from different host species. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced protective antibodies cross-reactive to antigenically different influenza A viruses and conferred cross protection. Anti-M2e antibodies induced by heterologous M2e5x VLPs showed a wider range of cross reactivity to influenza A viruses at higher levels than those by live virus infection, homologous M2e VLPs, or M2e monoclonal antibody 14C2. Fc receptors were found to be important for mediating protection by immune sera from M2e5x VLP vaccination. The present study provides evidence that heterologous recombinant M2e5x VLPs can be more effective in inducing protective M2e immunity than natural virus infection and further supports an approach for developing an effective universal influenza vaccine. Copyright © 2013 Elsevier B.V. All rights reserved.
Binley, Katie; Widdowson, Peter; Loader, Julie; Kelleher, Michelle; Iqball, Sharifah; Ferrige, Georgina; de Belin, Jackie; Carlucci, Marie; Angell-Manning, Diana; Hurst, Felicity; Ellis, Scott; Miskin, James; Fernandes, Alcides; Wong, Paul; Allikmets, Rando; Bergstrom, Christopher; Aaberg, Thomas; Yan, Jiong; Kong, Jian; Gouras, Peter; Prefontaine, Annick; Vezina, Mark; Bussieres, Martin; Naylor, Stuart; Mitrophanous, Kyriacos A
StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.
Benjamin M Althouse
Full Text Available Isolations of sylvatic dengue-2 virus from mosquitoes, humans and non-human primates in Senegal show synchronized multi-annual dynamics over the past 50 years. Host demography has been shown to directly affect the period between epidemics in other pathogen systems, therefore, one might expect unsynchronized multi-annual cycles occurring in hosts with dramatically different birth rates and life spans. However, in Senegal, we observe a single synchronized eight-year cycle across all vector species, suggesting synchronized dynamics in all vertebrate hosts. In the current study, we aim to explore two specific hypotheses: 1 primates with different demographics will experience outbreaks of dengue at different periodicities when observed as isolated systems, and that coupling of these subsystems through mosquito biting will act to synchronize incidence; and 2 the eight-year periodicity of isolations observed across multiple primate species is the result of long-term cycling in population immunity in the host populations. To test these hypotheses, we develop a multi-host, multi-vector Susceptible, Infected, Removed (SIR model to explore the effects of coupling multiple host-vector systems of dengue virus transmission through cross-species biting rates. We find that under small amounts of coupling, incidence in the host species synchronize. Long-period multi-annual dynamics are observed only when prevalence in troughs reaches vanishingly small levels (< 10(-10, suggesting that these dynamics are inconsistent with sustained transmission in this setting, but are consistent with local dengue virus extinctions followed by reintroductions. Inclusion of a constant introduction of infectious individuals into the system causes the multi-annual periods to shrink, while the effects of coupling remain the same. Inclusion of a stochastic rate of introduction allows for multi-annual periods at a cost of reduced synchrony. Thus, we conclude that the eight-year period
Græsbøll, Kaare; Bødker, Rene; Enøe, Claes
Bluetongue is a disease of ruminants which reached Denmark in 2007. We present a process-based stochastic simulation model of vector-borne diseases, where host animals are not confined to a central geographic farm coordinate, but can be distributed onto pasture areas. Furthermore vectors fly freely...
Eftekhariyan Ghamsari, Mohammad Reza; Karimi, Farah; Mousavi Gargari, Seyed Latif; Hosseini Tafreshi, Seyed Ali; Salami, Seyed Alireza
Datura stramonium is a well-known medicinal plant, which is important for its alkaloids. There are intrinsic limitations for the natural production of alkaloids in plants; metabolic engineering methods can be effectively used to conquer these limitations. In order for this the genes involved in corresponding pathways need to be studied. Virus-Induced Gene Silencing is known as a functional genomics technique to knock-down expression of endogenous genes. In this study, we silenced phytoene desaturase as a marker gene in D. stramonium in a heterologous and homologous manner by tobacco-rattle-virus-based VIGS vectors. Recombinant TRV vector containing pds gene from D. stramonium (pTRV2-Dspds) was constructed and injected into seedlings. The plants injected with pTRV2-Dspds showed photobleaching 2 weeks after infiltration. Spectrophotometric analysis demonstrated that the amount of chlorophylls and carotenoids in leaves of the bleached plants decreased considerably compared to that of the control plants. Semi-Quantitative RT-PCR results also confirmed that the expression of pds gene in the silenced plants was significantly reduced in comparison with the control plants. The results showed that the viral vector was able to influence the levels of total alkaloid content in D. stramonium. Our results illustrated that TRV-based VIGS vectors are able to induce effective and reliable functional gene silencing in D. stramonium as an alternative tool for studying the genes of interest in this plant, such as the targeted genes in tropane alkaloid biosynthetic pathway. The present work is the first report of establishing VIGS as an efficient method for transient silencing of any gene of interest in D. stramonium.
Miesbach, Wolfgang; Meijer, Karina; Coppens, Michiel
Hemophilia B gene therapy aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving...... expression of a codon-optimized wild-type human FIX gene. This multi-national, open-label study included ten adults with hemophilia B (FIX ≤2% of normal) and severe-bleeding phenotype. No participants tested positive for AAV5-neutralizing antibodies using a green-fluorescent protein-based assay and all 10...
Full Text Available Severe fever with thrombocytopenia syndrome virus (SFTSV, the causative agent for the fatal life-threatening infectious disease, severe fever with thrombocytopenia syndrome (SFTS, was first identified in the central and eastern regions of China. Although the viral RNA was detected in free-living and parasitic ticks, the vector for SFTSV remains unsettled.Firstly, an experimental infection study in goats was conducted in a bio-safety level-2 (BSL-2 facility to investigate virus transmission between animals. The results showed that infected animals did not shed virus to the outside through respiratory or digestive tract route, and the control animals did not get infected. Then, a natural infection study was carried out in the SFTSV endemic region. A cohort of naïve goats was used as sentinel animals in the study site. A variety of daily samples including goat sera, ticks and mosquitoes were collected for viral RNA and antibody (from serum only detection, and virus isolation. We detected viral RNA from free-living and parasitic ticks rather than mosquitoes, and from goats after ticks' infestation. We also observed sero-conversion in all members of the animal cohort subsequently. The S segment sequences of the two recovered viral isolates from one infected goat and its parasitic ticks showed a 100% homology at the nucleic acid level.In our natural infection study, close contact between goats does not appear to transmit SFTSV, however, the naïve animals were infected after ticks' infestation and two viral isolates derived from an infected goat and its parasitic ticks shared 100% of sequence identity. These data demonstrate that the etiologic agent for goat cohort's natural infection comes from environmental factors. Of these, ticks, especially the predominant species Haemaphysalis longicornis, probably act as vector for this pathogen. The findings in this study may help local health authorities formulate and focus preventive measures to contain
Full Text Available Eastern equine encephalitis virus (EEEV causes a highly pathogenic mosquito-borne zoonosis that is responsible for sporadic outbreaks of severe illness in humans and equines in the eastern USA. Culiseta (Cs. melanura is the primary vector of EEEV in most geographic regions but its feeding patterns on specific avian and mammalian hosts are largely unknown in the mid-Atlantic region. The objectives of our study were to: 1 identify avian hosts of Cs. melanura and evaluate their potential role in enzootic amplification of EEEV, 2 assess spatial and temporal patterns of virus activity during a season of intense virus transmission, and 3 investigate the potential role of Cs. melanura in epidemic/epizootic transmission of EEEV to humans and equines. Accordingly, we collected mosquitoes at 55 sites in Suffolk, Virginia in 2013, and identified the source of blood meals in engorged mosquitoes by nucleotide sequencing PCR products of the mitochondrial cytochrome b gene. We also examined field-collected mosquitoes for evidence of infection with EEEV using Vector Test, cell culture, and PCR. Analysis of 188 engorged Cs. melanura sampled from April through October 2013 indicated that 95.2%, 4.3%, and 0.5% obtained blood meals from avian, mammalian, and reptilian hosts, respectively. American Robin was the most frequently identified host for Cs. melanura (42.6% of blood meals followed by Northern Cardinal (16.0%, European Starling (11.2%, Carolina Wren (4.3%, and Common Grackle (4.3%. EEEV was detected in 106 mosquito pools of Cs. melanura, and the number of virus positive pools peaked in late July with 22 positive pools and a Maximum Likelihood Estimation (MLE infection rate of 4.46 per 1,000 mosquitoes. Our findings highlight the importance of Cs. melanura as a regional EEEV vector based on frequent feeding on virus-competent bird species. A small proportion of blood meals acquired from mammalian hosts suggests the possibility that this species may
Guo, Honggang; Wan, Shifan; Ge, Feng
Climatic variations are becoming important limiting factors for agriculture productivity, as they not only directly affect the plant net primary productivity but can also modulate the outbreak of plant diseases and pests. Elevated CO2 and O3 are two important climatic factors that have been widely studied before. Elevated CO2 or O3 alters the host plant physiology and affects the vector insects and plant viruses via bottom-up effects of the host plants. Many studies have shown that elevated CO2 or O3 decreases the plant nitrogen content, which modulates the characteristics of vector insects. Recent evidence also reveals that hormone-dependent signaling pathways play a critical role in regulating the response of insects and plant viruses to elevated CO2 or O3. In the current review, we describe how elevated CO2 or O3 affects the vector insects and plant viruses by altering the SA and JA signaling pathways. We also discuss how changes in the feeding behavior of vector insects or the occurrence of plant viruses affects the interactions between vector insects and plant viruses under elevated CO2 or O3. We suggest that new insights into the upstream network that regulates hormone signaling and top-down effects of natural enemies would provide a comprehensive understanding of the complex interactions taking place under elevated CO2 or O3.
Lehmann, Kathrin; Werner, Doreen; Hoffmann, Bernd; Kampen, Helge
Biting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV) in northern and central Europe during the 2006 outbreak of bluetongue disease (BT). Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV) in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI) gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus) plus the isomorphic species C. dewulfi. Biting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer. The newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic band for each species, could be realized with high
CHEN, SIFENG; KAPTURCZAK, MATTHIAS; LOILER, SCOTT A.; ZOLOTUKHIN, SERGEI; GLUSHAKOVA, OLENA Y.; MADSEN, KIRSTEN M.; SAMULSKI, RICHARD J.; HAUSWIRTH, WILLIAM W.; CAMPBELL-THOMPSON, MARTHA; BERNS, KENNETH I.; FLOTTE, TERENCE R.; ATKINSON, MARK A.; TISHER, C. CRAIG
Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies. OVERVIEW SUMMARY Gene delivery to the vasculature has significant potential as a therapeutic strategy for several cardiovascular disorders including atherosclerosis, hypertension, angiogenesis, and chronic vascular rejection of transplanted organs. However, limited advances have been made in achieving successful vascular endothelial cell gene transfer. The results of the present study demonstrate the superior efficacy of recombinant adeno-associated virus (rAAV) serotype 1 and 5 vectors in comparison to the traditionally used rAAV serotype 2 in transduction of primary vascular endothelial and smooth muscle cells in vitro. Our results have identified sialic acid residues for rAAV1 transduction in endothelial
Full Text Available Abstract Background Biting midges of the Obsoletus species complex of the ceratopogonid genus Culicoides were assumed to be the major vectors of bluetongue virus (BTV in northern and central Europe during the 2006 outbreak of bluetongue disease (BT. Most recently, field specimens of the same group of species have also been shown to be infected with the newly emerged Schmallenberg virus (SBV in Europe. A reliable identification of the cryptic species of this group is fundamental for both understanding the epidemiology of the diseases and for targeted vector control. In the absence of classical morphological characters unambiguously identifying the species, DNA sequence-based tests have been established for the distinction of selected species in some parts of Europe. Since specificity and sensitivity of these tests have been shown to be in need of improvement, an alternative PCR assay targeting the mitochondrial cytochrome oxidase subunit I (COI gene was developed for the identification of the three Obsoletus complex species endemic to Germany (C. obsoletus, C. scoticus, C. chiopterus plus the isomorphic species C. dewulfi. Methods Biting midges of the genus Culicoides caught by UV light traps all over Germany were morphologically pre-identified to species or complex level. The COI region was amplified from their extracted DNA and sequenced. Final species assignment was done by sequence comparison to GenBank entries and to morphologically identified males. Species-specific consensus sequences were aligned and polymorphisms were utilized to design species-specific primers to PCR-identify specimens when combined with a universal primer. Results The newly developed multiplex PCR assay was successfully tested on genetically defined Obsoletus complex material as well as on morphologically pre-identified field material. The intended major advantage of the assay as compared to other PCR approaches, namely the production of only one single characteristic
Killiny, N; Harper, S J; Alfaress, S; El Mohtar, C; Dawson, W O
Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-β-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere
Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E
Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.
Okada, Takashi; Nonaka-Sarukawa, Mutsuko; Uchibori, Ryosuke; Kinoshita, Kazue; Hayashita-Kinoh, Hiromi; Nitahara-Kasahara, Yuko; Takeda, Shin'ichi; Ozawa, Keiya
In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.
Nygårdas, Michaela; Paavilainen, Henrik; Müther, Nadine; Nagel, Claus-Henning; Röyttä, Matias; Sodeik, Beate; Hukkanen, Veijo
Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17(+))-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE.
Nyari, N; Maan, H S; Sharma, S; Pandey, S N; Dhole, T N
Chikungunya fever is an emerging mosquito-borne disease caused by the infection with chikungunya virus (CHIKV). The CHIKV has been rarely detected in mosquito vectors from Northern India, since vector surveillance is an effective strategy in controlling and preventing CHIKV transmission. Thus, virological investigation for CHIKV among mosquitoes of Aedes (A.) species was carried out in the Lucknow district during March 2010 to October 2011. We collected adult mosquitoes from areas with CHIKV positive patients. The adult Aedes mosquito samples were pooled, homogenized, clarified and tested for CHIKV by nonstructural protein 1 (nsP1) gene based polymerase chain reaction (PCR). A total 91 mosquito pools comprising of adult A. aegypti and A. albopictus were tested for CHIKV. The partial envelope protein (E1) gene sequences of mosquito-borne CHIKV strains were analyzed for genotyping. Of 91 pools, 6 pools of A. aegypti; and 2 pools of A. albopictus mosquitoes were identified positive for CHIKV by PCR. The phylogenetic analysis revealed clustering of CHIKV strains in two sub-lineages within the monophyletic East-Central South African (ECSA) genotype. Novel amino acid changes at the positions 294 (P294L) and 295 (S295F) were observed during analysis of amino acid sequence of the partial E1 gene. This study demonstrates the genetic diversity of circulating CHIKV strains and reports the first detection of CHIKV strains in Aedes vector species from the state of Uttar Pradesh. These findings have implication for vector control strategies to mitigate vector population to prevent the likelihood of CHIKV epidemic in the near future. Copyright © 2016 Elsevier B.V. All rights reserved.
Stable flies, which are notorious pests of cattle and other livestock, were suspected of transmitting West Nile virus (WNV) among American white pelicans at the Medicine Lake Wildlife Refuge in northeastern Montana in 2006-2007. However the ability of stable flies to transmit the virus was unknown. ...
Bernards, R.A.; Destree, A.; McKenzie, S.; Gordon, E.; Weinberg, R.A.; Panicali, D.
We have constructed a vaccinia virus recombinant that expresses the extracellular domain of the rat neu oncogene-encoded protein, a 185-kDa transmembrane glycoprotein termed p185. Strain NFS mice immunized with this recombinant virus developed a strong antibody response against the neu oncogene
Kamencayová, M; Košík, I; Hunková, J; Subr, Z W
PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.
The purpose of the proposed research is to develop a propagation-competent (PC) aiphavirus vector that is targeted specifically to receptors expressed on breast cancer cells or to receptors expressed on tumor-associated vasculature...
Cenna, Jonathan; Tan, Gene S; Papaneri, Amy B; Dietzschold, Bernhard; Schnell, Matthias J; McGettigan, James P
The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-DeltaP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-DeltaP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-DeltaP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a 10-fold higher efficiency of live SPBN-DeltaP compared to UV-inactivated SPBN-DeltaP. In addition, SPBN-DeltaP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-DeltaP. Of note, 10(3)ffu of SPBN-DeltaP-RVG-induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-DeltaP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 10(5)ffu/mouse of SPBN-DeltaP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune
Kawagoe, Shiho; Higuchi, Takashi; Otaka, Manami; Shimada, Yohta; Kobayashi, Hiroshi; Ida, Hiroyuki; Ohashi, Toya; Okano, Hirotaka J; Nakanishi, Mahito; Eto, Yoshikatsu
We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture. Copyright © 2013 Elsevier Inc. All rights reserved.
Lim, Seungmo; Nam, Moon; Kim, Kil Hyun; Lee, Su-Heon; Moon, Jung-Kyung; Lim, Hyoun-Sub; Choung, Myoung-Gun; Kim, Sang-Mok; Moon, Jae Sun
A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C
Galal Fatma H
Full Text Available Abstract Background Rift Valley fever (RVF is an acute febrile arthropod-borne viral disease of man and animals caused by a member of the Phlebovirus genus, one of the five genera in the family Bunyaviridae. RVF virus (RVFV is transmitted between animals and human by mosquitoes, particularly those belonging to the Culex, Anopheles and Aedes genera. Methods Experiments were designed during RVF outbreak, 2007 in Sudan to provide an answer about many raised questions about the estimated role of vector in RVFV epidemiology. During this study, adult and immature mosquito species were collected from Khartoum and White Nile states, identified and species abundance was calculated. All samples were frozen individually for further virus detection. Total RNA was extracted from individual insects and RVF virus was detected from Culex, Anopheles and Aedes species using RT-PCR. In addition, data were collected about human cases up to November 24th, 2007 to asses the situation of the disease in affected states. Furthermore, a historical background of the RVF outbreaks was discussed in relation to global climatic anomalies and incriminated vector species. Results A total of 978 mosquitoes, belonging to 3 genera and 7 species, were collected during Sudan outbreak, 2007. Anopheles gambiae arabiensis was the most frequent species (80.7% in White Nile state. Meanwhile, Cx. pipiens complex was the most abundant species (91.2% in Khartoum state. RT-PCR was used and successfully amplified 551 bp within the M segment of the tripartite negative-sense single stranded RNA genome of RVFV. The virus was detected in female, male and larval stages of Culex and Anopheles species. The most affected human age interval was 15-29 years old followed by ≥ 45 years old, 30-44 years old, and then 5-14 years old. Regarding to the profession, housewives followed by farmers, students, shepherd, workers and the free were more vulnerable to the infection. Furthermore, connection between
Yan, Jingyi; Dong, Jianing; Wu, Jiaxin; Zhu, Rui; Wang, Zhen; Wang, Baoming; Wang, Lizheng; Wang, Zixuan; Zhang, Haihong; Wu, Hui; Yu, Bin; Kong, Wei; Yu, Xianghui
The immunogenicity of recombinant adenovirus serotype 5 (rAd5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against hexon hypervariable regions (HVRs). Preexisting immunity can be circumvented by replacing HVRs of rAd5 hexon with those derived from alternate adenovirus serotypes. However, chimeric modification of rAd5 hexon HVRs tends to cause low packaging efficiency or low proliferation of rAd5 vectors, but the related mechanism remains unclear. In this study, several Ad5-based vectors with precise replacement of HVRs with those derived from Ad37 and Ad43 were generated. We first observed that a HVR-exchanged rAd5 vector displayed a higher efficacy of the recombinant virus rescue and growth improvement compared with the rAd5 vector, although most hexon-chimeric rAd5 vectors constructed by us and other groups have proven to be nonviable or growth defective. We therefore evaluated the structural stability of the chimeric hexons and their interactions with the L4-100K chaperone. We showed that the viability of hexon-chimeric Ad5 vectors was not attributed to the structural stability of the chimeric hexon, but rather to the hexon maturation which was assisted by L4-100K. Our results suggested that the intricate interaction between hexon and L4-100K would determine the virus rescue and proliferation efficiency of hexon-chimeric rAd5 vectors.
Joanne, Sylvia; Vythilingam, Indra; Teoh, Boon-Teong; Leong, Cherng-Shii; Tan, Kim-Kee; Wong, Meng-Li; Yugavathy, Nava; AbuBakar, Sazaly
To determine the susceptibility status of Aedes albopictus with and without Wolbachia to the four dengue virus serotypes. Two newly colonised colonies of Ae. albopictus from the wild were used for the study. One colony was naturally infected with Wolbachia while in the other Wolbachia was removed by tetracycline treatment. Both colonies were orally infected with dengue virus-infected fresh blood meal. Dengue virus load was measured using quantitative RT-PCR at four-time intervals in the salivary glands, midguts and ovaries. Wolbachia did not significantly affect Malaysian Ae. albopictus dengue infection or the dissemination rate for all four dengue virus serotypes. Malaysian Ae. albopictus had the highest replication kinetics for DENV-1 and the highest salivary gland and midgut infection rate for DENV-4. Wolbachia, which naturally exists in Malaysian Ae. albopictus, does not significantly affect dengue virus replication. Malaysian Ae. albopictus is susceptible to dengue virus infections and capable of transmitting dengue virus, especially DENV-1 and DENV-4. Removal of Wolbachia from Malaysian Ae. albopictus would not reduce their susceptibility status. © 2017 John Wiley & Sons Ltd.
Full Text Available Recombinant adeno-associated viruses (rAAV are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.
Wallis, C M; Fleischer, S J; Luster, D; Gildow, F E
Plum pox, an invasive disease recently identified in Pennsylvania stone fruit orchards, is caused by the aphid-transmitted Plum pox virus (genus Potyvirus, family Potyviridae, PPV). To identify potential vectors, we described the aphid species communities and the seasonal dynamics of the dominant aphid species within Pennsylvania peach orchards. Aphids were trapped weekly in 2002 and 2003 from mid-April through mid-November within two central Pennsylvania orchards by using yellow and green water pan traps. In total, 42 aphid species were identified from both orchards over 2 yr. Within orchards, actual species richness ranged from 24 to 30 species. The Abundance Based Coverage Estimator predicted species richness to range from 30 to 36 species, indicating that trap catches were identifying most aphid species expected to occur in the orchard. Three species, Rhopalosiphum maidis (Fitch), Aphis spiraecola Patch, and Myzus persicae (Sulzer), were consistently dominant across locations and years. Orchard-trapped populations of these three species peaked in a similar chronological sequence each year. As expected, trap color influenced the total number and distribution of the predominate species collected. However, the same dominant species occurred in both yellow and green traps. Based on the seasonal population dynamics reported here and on published vector efficacy studies, the most probable significant PPV vector was identified as A. spiraecola. If the PPV pathogen escapes current quarantine or if subsequent reintroductions of PPV occur, these data will be useful for developing plum pox management strategies.
Full Text Available Abstract Background Gene silencing vectors based on Barley stripe mosaic virus (BSMV are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species have created a need for tools to study gene function in these species. Results Here we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa and diploid (A. strigosa oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component. Conclusions Our results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too
Full Text Available Feeding success of free-living hematophagous insects depends on their ability to be active when hosts are available and to reach places where hosts are accessible. When the hematophagous insect is a vector of pathogens, determining the components of host-seeking behavior is of primary interest for the assessment of transmission risk. Our aim was to describe endo/exophagy and circadian host-seeking activity of Palaearctic Culicoides species, which are major biting pests and arbovirus vectors, using drop traps and suction traps baited with four sheep, as bluetongue virus hosts. Collections were carried out in the field, a largely-open stable and an enclosed stable during six collection periods of 24 hours in April/May, in late June and in September/October 2010 in western France. A total of 986 Culicoides belonging to 13 species, mainly C. brunnicans and C. obsoletus, was collected on animal baits. Culicoides brunnicans was clearly exophagic, whereas C. obsoletus was able to enter stables. Culicoides brunnicans exhibited a bimodal pattern of host-seeking activity with peaks just after sunrise and sunset. Culicoides obsoletus was active before sunset in spring and autumn and after sunset in summer, thus illustrating influence of other parameters than light, especially temperature. Description of host-seeking behaviors allowed us to discuss control strategies for transmission of Culicoides-borne pathogens, such as bluetongue virus. However, practical vector-control recommendations are difficult to provide because of the variation in the degree of endophagy and time of host-seeking activity.
Full Text Available Foamy virus (FV vectors are promising tools for gene therapy, but low titer is a major challenge for large-scale clinical trials. Here, we increased FV vector titer 50-fold by constructing novel vector plasmids and using polyethylenimine-mediated transfection. FV and lentiviral (LV vectors were used separately to transduce human CD34+ cells at multiplicities of infection of 25, and those cells were transplanted into immunodeficient mice. FV vector transduction frequencies of repopulating human cells were 37.1 ± 1.9% in unstimulated cells and 36.9 ± 2.2% in prestimulated cells, and engraftment frequencies were 40.9 ± 4.9% in unstimulated cells and 47.1 ± 3.3% in prestimulated cells. Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells. Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus. FV had an integration preference near transcriptional start sites and CpG islands of RefSeq genes but not within genes. Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations. Our new FV vector backbone may be a suitable candidate for developing therapeutic FV vectors for use in clinical trials.
Bostan, Nazish; Javed, Sundus; Nabgha-E-Amen; Eqani, Syed Ali Musstjab Akber Shah; Tahir, Faheem; Bokhari, Habib
Dengue fever is regarded as one of the most prominent emerging arboviral infections in Pakistan since its first epidemic almost 2 decades ago. Interplay between potential vectors, susceptible host, and lax environmental conditions may promote the infection, leading to an epidemic. These factors may indeed have played a major role in the spread of the disease in the country, which was limited to Karachi till 2006. With recent natural disasters such as the earthquake in 2005 and flooding in 2010, 2011 and 2012, numbers of vector-borne diseases and outbreaks including dengue fever are on the rise in Pakistan. Therefore, it is a major concern for health sector workers and of utmost importance to have some understanding of the factors affecting disease outbreak for better risk assessment in the region. In the following report we review the climatic as well as host- and vector-associated factors involved in the outbreak of dengue epidemics in Pakistan and highlight high-risk zones in the country. Copyright © 2016 John Wiley & Sons, Ltd.
Duraffour, S.; Andrei, G.; Topalis, D.; Krečmerová, Marcela; Crance, J. M.; Garin, D.; Snoeck, R.
Roč. 86, č. 13 (2012), s. 7310-7325 ISSN 0022-538X Institutional support: RVO:61388963 Keywords : camelpox virus * CMLV * vaccinia virus VACV * acyclic nucleoside phosphonates * HPMPDAP * cidofovir * drug resistance Subject RIV: CC - Organic Chemistry Impact factor: 5.076, year: 2012
Zhao, Fumei; Lim, Seungmo; Igori, Davaajargal; Yoo, Ran Hee; Kwon, Suk-Yoon; Moon, Jae Sun
We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR. Copyright © 2016 Elsevier Inc. All rights reserved.
Cors, Josep; Llibre, Jaume; Korobeinikov, Andrei
The two parts of the present volume contain extended conference abstracts corresponding to selected talks given by participants at the "Conference on Hamiltonian Systems and Celestial Mechanics 2014" (HAMSYS2014) (15 abstracts) and at the "Workshop on Virus Dynamics and Evolution" (12 abstracts), both held at the Centre de Recerca Matemàtica (CRM) in Barcelona from June 2nd to 6th, 2014, and from June 23th to 27th, 2014, respectively. Most of them are brief articles, containing preliminary presentations of new results not yet published in regular research journals. The articles are the result of a direct collaboration between active researchers in the area after working in a dynamic and productive atmosphere. The first part is about Central Configurations, Periodic Orbits and Hamiltonian Systems with applications to Celestial Mechanics – a very modern and active field of research. The second part is dedicated to mathematical methods applied to viral dynamics and evolution. Mathematical modelling of biologi...
Mardulyn, Patrick; Goffredo, Maria; Conte, Annamaria; Hendrickx, Guy; Meiswinkel, Rudolf; Balenghien, Thomas; Sghaier, Soufien; Lohr, Youssef; Gilbert, Marius
Bluetongue (BT) is a commonly cited example of a disease with a distribution believed to have recently expanded in response to global warming. The BT virus is transmitted to ruminants by biting midges of the genus Culicoides, and it has been hypothesized that the emergence of BT in Mediterranean Europe during the last two decades is a consequence of the recent colonization of the region by Culicoides imicola and linked to climate change. To better understand the mechanism responsible for the northward spread of BT, we tested the hypothesis of a recent colonization of Italy by C. imicola, by obtaining samples from more than 60 localities across Italy, Corsica, Southern France, and Northern Africa (the hypothesized source point for the recent invasion of C. imicola), and by genotyping them with 10 newly identified microsatellite loci. The patterns of genetic variation within and among the sampled populations were characterized and used in a rigorous approximate Bayesian computation framework to compare three competing historical hypotheses related to the arrival and establishment of C. imicola in Italy. The hypothesis of an ancient presence of the insect vector was strongly favoured by this analysis, with an associated P ≥ 99%, suggesting that causes other than the northward range expansion of C. imicola may have supported the emergence of BT in southern Europe. Overall, this study illustrates the potential of molecular genetic markers for exploring the assumed link between climate change and the spread of diseases. © 2013 Blackwell Publishing Ltd.
Maris, P.C.; Joosten, N.N.; Goldbach, R.W.; Peters, D.
The effect of Tomato spotted wilt virus (TSWV) infection on plant attractiveness for the western flower thrips (Frankliniella occidentalis) was studied. Significantly more thrips were recovered on infected than were recovered on noninfected pepper (Capsicum annuum) plants in different preference
Zhang, H Steve; Kim, Eunmi; Lee, Slgirim; Ahn, Ik-Sung; Jang, Jae-Hyung
Recombinant adeno-associated virus (AAV) vectors can be engineered to carry genetic material encoding therapeutic gene products that have demonstrated significant clinical promise. These viral vectors are typically produced in mammalian cells by the transient transfection of two or three plasmids encoding the AAV rep and cap genes, the adenovirus helper gene, and a gene of interest. Although this method can produce high-quality AAV vectors when used with multiple purification protocols, one critical limitation is the difficulty in scaling-up manufacturing, which poses a significant hurdle to the broad clinical utilization of AAV vectors. To address this challenge, recombinant herpes simplex virus type I (rHSV-1)- and recombinant baculovirus (rBac)-based methods have been established recently. These methods are more amenable to large-scale production of AAV vectors than methods using the transient transfection of mammalian cells. To investigate potential applications of AAV vectors produced by rHSV-1- or rBac-based platforms, the in vivo transduction of rHSV-1- or rBac-produced AAV serotype 2 (AAV2) vectors within the rat brain were examined by comparing them with vectors generated by the conventional transfection method. Injection of rHSV-1- or rBac-produced AAV vectors into rat striatum and cortex tissues revealed no differences in cellular tropism (i.e., predominantly neuronal targeting) or anteroposterior spread compared with AAV2 vectors produced by transient transfection. This report represents a step towards validating AAV vectors produced by the rHSV-1- and the rBac-based systems as promising tools, especially for delivering therapeutic molecules to the central nervous system. Copyright © 2011 Elsevier B.V. All rights reserved.
You, H; Liu, Y; Cong, M; Ping, W; You, C; Zhang, D; Mehta, J L; Hermonat, P L
Hepatitis B virus (HBV) has been an increasing problem throughout the world and remains difficult to treat. But immunotherapeutic approaches offer new, effective treatments. Three recombinant adeno-associated virus (AAV) type 2 vectors, carrying one of the HBV S, C or X gene, were used to load (transduce) professional antigen-presenting dendritic cells (DC) for the purpose of stimulating cytotoxic T lymphocytes (CTL) in vitro. It was found that all three recombinant AAV/HBV antigen virus loaded DC at approximately 90% transduction efficiency. Most importantly, all three AAV-loaded DC stimulated rapid, antigen-specific and major histocompatibility complex (MHC)-restricted CTL. In vitro, these CTL killed (30-50%) synthetic antigen-positive autologous targets as well as HepG2 liver cell targets. In comparing the three antigens, it was found that AAV/HBV-C-derived CTL consistently had the highest killing efficiency. CTL derived from AAV/HBV-C-loaded DC also showed significantly higher killing of targets than that from bacterially generated C-protein-loaded DC. Further studies showed that AAV/HBV-C-derived CTL had higher interferon (IFN)-gamma. These data suggest that AAV/HBV antigen gene-loading of DC may be useful for immunotherapeutic protocols against HBV infection and that the HBV C antigen may be the most useful for this purpose.
Bricault, Christine A. [Department of Plant Pathology and Plant-Microbe Biology, 334 Plant Science Building, Cornell University, Ithaca, NY 14850 (United States); Perry, Keith L., E-mail: KLP3@cornell.edu [Department of Plant Pathology and Plant-Microbe Biology, 334 Plant Science Building, Cornell University, Ithaca, NY 14850 (United States)
In the atomic model of Cucumber mosaic virus (CMV), six amino acid residues form stabilizing salt bridges between subunits of the asymmetric unit at the quasi-threefold axis of symmetry. To evaluate the effects of these positions on virion stability and aphid vector transmissibility, six charged amino acid residues were individually mutated to alanine. All of the six engineered viruses were viable and exhibited near wild type levels of virion stability in the presence of urea. Aphid vector transmissibility was nearly or completely eliminated in the case of four of the mutants; two mutants demonstrated intermediate aphid transmissibility. For the majority of the engineered mutants, second-site mutations were observed following aphid transmission and/or mechanical passaging, and one restored transmission rates to that of the wild type. CMV capsids tolerate disruption of acid–base pairing interactions at the quasi-threefold axis of symmetry, but these interactions are essential for maintaining aphid vector transmissibility. - Highlights: ► Amino acids between structural subunits of Cucumber mosaic virus affect vector transmission. ► Mutant structural stability was retained, while aphid vector transmissibility was disrupted. ► Spontaneous, second-site mutations restored aphid vector transmissibility.
Chen, Sifeng; Kapturczak, Matthias; Loiler, Scott A; Zolotukhin, Sergei; Glushakova, Olena Y; Madsen, Kirsten M; Samulski, Richard J; Hauswirth, William W; Campbell-Thompson, Martha; Berns, Kenneth I; Flotte, Terence R; Atkinson, Mark A; Tisher, C Craig; Agarwal, Anupam
Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.
van Dodewaard, Caitlin A M; Richards, Stephanie L; Harris, Jonathan W
Commercially available blood can be used as an alternative to live animals to maintain mosquito colonies and deliver infectious bloodmeals during research studies. We analyzed the extent to which two methods for blood coagulate removal (defibrination or addition of sodium citrate) affected life table characteristics (i.e., fecundity, fertility, hatch rate, and adult survival) and vector competence (infection, dissemination, and transmission) of Aedes albopictus (Skuse) for dengue virus (DENV). Two types of bovine blood were tested at two extrinsic incubation temperatures (27 or 30°C) for DENV-infected and uninfected mosquitoes. Fully engorged mosquitoes were transferred to individual cages containing an oviposition cup and a substrate. Eggs (fecundity) and hatched larvae (fertility) were counted. At 14 and 21 d post feeding on a DENV-infected bloodmeal, 15 mosquitoes were sampled from each group, and vector competence was analyzed (bodies [infection], legs [dissemination], and saliva [transmission]). Differences in life table characteristics and vector competence were analyzed for mosquitoes fed blood processed using different methods for removal of coagulates. The method for removal of coagulates significantly impacted fecundity, fertility, and hatch time in the uninfected group, but not DENV-infected group. Infected mosquitoes showed significantly higher fecundity and faster hatch time than uninfected mosquitoes. We show no significant differences in infection or dissemination rates between groups; however, horizontal transmission rate was significantly higher in mosquitoes fed DENV-infected citrated compared with defibrinated blood. We expect the findings of this study to inform research using artificial blood delivery methods to assess vector competence. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: firstname.lastname@example.org.
Passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity.
Osorio, F A; Galeota, J A; Nelson, E; Brodersen, B; Doster, A; Wills, R; Zuckermann, F; Laegreid, W W
Immune mechanisms mediating protective immunity against porcine reproductive and respiratory syndrome virus (PRRSV) are not well understood. The PRRSV-specific humoral immune response has been dismissed as being ineffective and perhaps deleterious for the host. The function of PRRSV antibodies in protective immunity against infection with a highly abortifacient strain of this virus was examined by passive transfer experiments in pregnant swine. All of a group of pregnant gilts (n = 6) that received PRRSV immunoglobulin (Ig) from PRRSV-convalescent, hyperimmune animals were fully protected from reproductive failure as judged by 95% viability of offspring at weaning (15 days of age). On the other hand, the totality of animals in a matched control group (n = 6) receiving anti-pseudorabies virus (PRV) Ig exhibited marked reproductive failure with 4% survival at weaning. Besides protecting the pregnant females from clinical reproductive disease, the passive transfer of PRRSV Ig prevented the challenge virus from infecting the dams and precluded its vertical transmission, as evidenced by the complete absence of infectious PRRSV from the tissues of the dams and lack of infection in their offspring. In summary, these results indicate that PRRSV-Igs are capable of conferring protective immunity against PRRSV and furthermore that these Igs can provide sterilizing immunity in vivo.
Basner-Tschakarjan, Etiena; Bijjiga, Enoch; Martino, Ashley T
Transitioning to human trials from pre-clinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity and explored methods for bypassing these responses. Many efforts toward measuring innate immunity have utilized Toll-like receptor deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T-cells as well as cytotoxicity toward AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T-cell proliferation, but actual transgene level reduction and parameters of cytotoxicity toward transduced target cells have only been shown in one model. The model utilized adoptive transfer of capsid-specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells as well as modulating the response pharmacologically has also
Hareendran, Sangeetha; Balakrishnan, Balaji; Sen, Dwaipayan; Kumar, Sanjay; Srivastava, Alok; Jayandharan, Giridhara R
AAV-based gene transfer protocols have shown remarkable success when directed to immune-privileged sites such as for retinal disorders like Lebers congenital amaurosis. In contrast, AAV-mediated gene transfer into liver or muscle tissue for diseases such as hemophilia B, α1 anti-trypsin deficiency and muscular dystrophy has demonstrated a decline in gene transfer efficacy over time. It is now known that in humans, AAV triggers specific pathways that recruit immune sensors. These factors initiate an immediate reaction against either the viral capsid or the vector encoded protein as part of innate immune response or to produce a more specific adaptive response that generates immunological memory. The vector-transduced cells are then rapidly destroyed due to this immune activation. However, unlike other viral vectors, AAV is not immunogenic in murine models. Its immunogenicity becomes apparent only in large animal models and human subjects. Moreover, humans are natural hosts to AAV and exhibit a high seroprevalence against AAV vectors. This limits the widespread application of AAV vectors into patients with pre-existing neutralising antibodies or memory T cells. To address these issues, various strategies are being tested. Alternate serotype vectors (AAV1-10), efficient expression cassettes, specific tissue targeting, immune-suppression and engineered capsid variants are some approaches proposed to minimise this immune stimulation. In this review, we have summarised the nature of the immune response documented against AAV in various pre-clinical and clinical settings and have further discussed the strategies to evade them. Copyright © 2013 John Wiley & Sons, Ltd.
Full Text Available Transitioning to human trials from preclinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity, and explored methods for bypassing these responses. Many efforts towards measuring innate immunity have utilized Toll-Like Receptor (TLR deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody (NAb assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T cells as well as cytotoxicity towards AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T cell proliferation, but actual transgene level reduction and parameters of cytotoxicity towards transduced target cells has only been shown in one model. The model utilized adoptive transfer of capsid specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells has also been explored as well
Chimeric avian paramyxovirus-based vector immunization against highly pathogenic avian influenza followed by conventional Newcastle disease vaccination eliminates lack of protection from virulent ND virus
Steglich, C.; Grund, C.; A. Röder; Zhao, N.; Mettenleiter, T C; Römer-Oberdörfer, A.
Recently, we described a chimeric, hemagglutinin of highly pathogenic avian influenza virus (HPAIV) H5 expressing Newcastle disease virus (NDV)-based vector vaccine (chNDVFHNPMV8H5) in which NDV envelope glycoproteins were replaced by those of avian paramyxovirus-8 (APMV-8). This chimeric vaccine induced solid protection against lethal HPAIV H5N1 even in chickens with maternal antibodies against NDV (MDA+). However, due to the absence of the major NDV immunogens it failed to induce protection...
Full Text Available Abstract Background Culex spp. mosquitoes are considered to be the most important vectors of West Nile virus (WNV detected in at least 34 species of mosquitoes in the United States. In North America, Culex pipiens pipiens, Culex pipiens quinquefasciatus, and Culex tarsalis are all competent vectors of WNV, which is considered to be enzootic in the United States and has also been detected in equines and birds in many states of Mexico and in humans in Nuevo Leon. There is potential for WNV to be introduced into Mexico City by various means including infected mosquitoes on airplanes, migrating birds, ground transportation and infected humans. Little is known of the geographic distribution of Culex pipiens complex mosquitoes and hybrids in Mexico City. Culex pipiens pipiens preferentially feed on avian hosts; Culex pipiens quinquefasciatus have historically been considered to prefer mammalian hosts; and hybrids of these two species could theoretically serve as bridge vectors to transmit WNV from avian hosts to humans and other mammalian hosts. In order to address the potential of WNV being introduced into Mexico City, we have determined the identity and spatial distribution of Culex pipiens complex mosquitoes and their hybrids. Results Mosquito larvae collected from 103 sites throughout Mexico City during 2004-2005 were identified as Culex, Culiseta or Ochlerotatus by morphological analysis. Within the genus Culex, specimens were further identified as Culex tarsalis or as belonging to the Culex pipiens complex. Members of the Culex pipiens complex were separated by measuring the ratio of the dorsal and ventral arms (DV/D ratio of the male genitalia and also by using diagnostic primers designed for the Ace.2 gene. Culex pipiens quinquefasciatus was the most abundant form collected. Conclusions Important WNV vectors species, Cx. p. pipiens, Cx. p. quinquefasciatus and Cx. tarsalis, are all present in Mexico City. Hybrids of Cx. p. pipiens and Cx. p
Kang, Yun; Blanco, Krystal; Davis, Talia; Wang, Ying; DeGrandi-Hoffman, Gloria
The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses carried by Varroa mites. In this paper, we propose a honeybee-mite-virus model that incorporates (1) parasitic interactions between honeybees and the Varroa mites; (2) five virus transmission terms between honeybees and mites at different stages of Varroa mites: from honeybees to honeybees, from adult honeybees to the phoretic mites, from brood to the reproductive mites, from the reproductive mites to brood, and from adult honeybees to the phoretic mites; and (3) Allee effects in the honeybee population generated by its internal organization such as division of labor. We provide completed local and global analysis for the full system and its subsystems. Our analytical and numerical results allow us have a better understanding of the synergistic effects of parasitism and virus infections on honeybee population dynamics and its persistence. Interesting findings from our work include: (a) due to Allee effects experienced by the honeybee population, initial conditions are essential for the survival of the colony. (b) Low adult honeybees to brood ratios have destabilizing effects on the system which generate fluctuating dynamics that lead to a catastrophic event where both honeybees and mites suddenly become extinct. This catastrophic event could be potentially linked to Colony Collapse Disorder (CCD) of honeybee colonies. (c) Virus infections may have stabilizing effects on the system, and parasitic mites could make disease more persistent. Our model illustrates how the synergy between the parasitic mites and virus infections consequently generates rich dynamics including multiple attractors where all species can coexist or go extinct depending on initial conditions. Our findings may provide important insights on honeybee viruses and parasites and how to best control them
Davidson, Beverly L.; Stein, Colleen S.; Heth, Jason A.; Martins, Inês; Kotin, Robert M; Derksen, Todd A.; Zabner, Joseph; Ghodsi, Abdi; Chiorini, John A.
Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of β-galactosidase-encoding vectors made...
Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.
Sun, Kai; Zhao, Danyang; Liu, Yong; Huang, Changjun; Zhang, Wei; Li, Zhenghe
The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active circular DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast- Escherichia coli - Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.
Full Text Available The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.
Gillard, Geoffrey O.; Bivas-Benita, Maytal; Hovav, Avi-Hai; Grandpre, Lauren E.; Panas, Michael W.; Seaman, Michael S.; Haynes, Barton F.; Letvin, Norman L.
While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1+ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance. PMID:21829360
David P Tchouassi
Full Text Available Rift Valley fever (RVF outbreaks in Kenya have increased in frequency and range to include northeastern Kenya where viruses are increasingly being isolated from known (Aedes mcintoshi and newly-associated (Ae. ochraceus vectors. The factors contributing to these changing outbreak patterns are unclear and the population genetic structure of key vectors and/or specific virus-vector associations, in particular, are under-studied. By conducting mitochondrial and nuclear DNA analyses on >220 Kenyan specimens of Ae. mcintoshi and Ae. ochraceus, we uncovered high levels of vector complexity which may partly explain the disease outbreak pattern. Results indicate that Ae. mcintoshi consists of a species complex with one of the member species being unique to the newly-established RVF outbreak-prone northeastern region of Kenya, whereas Ae. ochraceus is a homogeneous population that appears to be undergoing expansion. Characterization of specimens from a RVF-prone site in Senegal, where Ae. ochraceus is a primary vector, revealed direct genetic links between the two Ae. ochraceus populations from both countries. Our data strongly suggest that unlike Ae. mcintoshi, Ae. ochraceus appears to be a relatively recent, single 'introduction' into Kenya. These results, together with increasing isolations from this vector, indicate that Ae. ochraceus will likely be of greater epidemiological importance in future RVF outbreaks in Kenya. Furthermore, the overall vector complexity calls into question the feasibility of mosquito population control approaches reliant on genetic modification.
Young, Katherine I; Mundis, Stephanie; Widen, Steven G; Wood, Thomas G; Tesh, Robert B; Cardosa, Jane; Vasilakis, Nikos; Perera, David; Hanley, Kathryn A
Mosquito-borne dengue virus (DENV) is maintained in a sylvatic, enzootic cycle of transmission between canopy-dwelling non-human primates and Aedes mosquitoes in Borneo. Sylvatic DENV can spill over into humans living in proximity to forest foci of transmission, in some cases resulting in severe dengue disease. The most likely vectors of such spillover (bridge vectors) in Borneo are Ae. albopictus and Ae. niveus. Borneo is currently experiencing extensive forest clearance. To gauge the effect of this change in forest cover on the likelihood of sylvatic DENV spillover, it is first necessary to characterize the distribution of bridge vectors in different land cover types. In the current study, we hypothesized that Ae. niveus and Ae. albopictus would show significantly different distributions in different land cover types; specifically, we predicted that Ae. niveus would be most abundant in forests whereas Ae. albopictus would have a more even distribution in the landscape. Mosquitoes were collected from a total of 15 sites using gravid traps and a backpack aspirator around Kampong Puruh Karu, Sarawak, Malaysian Borneo, where sylvatic DENV spillover has been documented. A total of 2447 mosquitoes comprising 10 genera and 4 species of Aedes, were collected over the three years, 2013, 2014 and 2016, in the three major land cover types in the area, homestead, agriculture and forest. Mosquitoes were identified morphologically, pooled by species and gender, homogenized, and subject to DNA barcoding of each Aedes species and to arbovirus screening. As predicted, Ae. niveus was found almost exclusively in forests whereas Ae. albopictus was collected in all land cover types. Aedes albopictus was significantly (P = 0.04) more abundant in agricultural fields than forests. Sylvatic DENV was not detected in any Aedes mosquito pools, however genomes of 14 viruses were detected using next generation sequencing. Land cover type affects the abundance and distribution of the most
Roberts, H.C.; Elbers, A.R.W.; Conraths, F.J.; Holsteg, M.; Hoereth-Boentgen, D.; Gethmann, J.; Schaik, van G.
Surveillance for new emerging animal diseases from a European perspective is complicated by the non-harmonised approach across Member States for data capture, recording livestock populations and case definitions. In the summer of 2011, a new vector-borne Orthobunyavirus emerged in Northern Europe
1972-01-01In search of the site of PLRV multiplication in its vector a detailed study was made of the anatomy of the aphid, Myzus persicae SULZ. The findings are summarized in the following lines:Alimentary canalThe most anterior part of
Cui, Hongguang; Wang, Aiming
RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Full Text Available Bovine viral diarrhea virus (BVDV plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2 to broaden coverage. BVDV modified live virus (MLV vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373 challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding
Amraoui, Fadila; Krida, Ghazi; Bouattour, Ali; Rhim, Adel; Daaboub, Jabeur; Harrat, Zoubir; Boubidi, Said-Chawki; Tijane, Mhamed; Sarih, Mhammed; Failloux, Anna-Bella
West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8) and 10(8.5) plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.
Full Text Available West Nile fever (WNF and Rift Valley fever (RVF are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8 and 10(8.5 plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.
Tamura, Rodrigo Esaki; da Silva Soares, Rafael Bento; Costanzi-Strauss, Eugenia; Strauss, Bryan E
Alternative treatments for cancer using gene therapy approaches have shown promising results and some have even reached the marketplace. Even so, additional improvements are needed, such as employing a strategically chosen promoter to drive expression of the transgene in the target cell. Previously, we described viral vectors where high-level transgene expression was achieved using a p53-responsive promoter. Here we present an adenoviral vector (AdPGp53) where p53 is employed to regulate its own expression and which outperforms a traditional vector when tested in a model of gene therapy for prostate cancer. The functionality of AdPGp53 and AdCMVp53 were compared in human prostate carcinoma cell lines. AdPGp53 conferred greatly enhanced levels of p53 protein and induction of the p53 target gene, p21, as well as superior cell killing by a mechanism consistent with apoptosis. DU145 cells were susceptible to induction of death with AdPGp53, yet PC3 cells were quite resistant. Though AdCMVp53 was shown to be reliable, extremely high-level expression of p53 offered by AdPGp53 was necessary for tumor suppressor activity in PC3 and DU145. In situ gene therapy experiments revealed tumor inhibition and increased overall survival in response to AdPGp53, but not AdCMVp53. Upon histologic examination, only AdPGp53 treatment was correlated with the detection of both p53 and TUNEL-positive cells. This study points to the importance of improved vector performance for gene therapy of prostate cancer.
Ngu, Loveline N; Nji, Nadesh N; Ambada, Georgia; Ngoh, Apeh A; Njambe Priso, Ghislain D; Tchadji, Jules C; Lissom, Abel; Magagoum, Suzanne H; Sake, Carol N; Tchouangueu, Thibau F; Chukwuma, George O; Okoli, Arinze S; Sagnia, Bertrand; Chukwuanukwu, Rebecca; Tebit, Denis M; Esimone, Charles O; Waffo, Alain B; Park, Chae G; Überla, Klaus; Nchinda, Godwin W
Recombinant Newcastle Disease virus (rNDV) vectored vaccines are safe mucosal applicable vaccines with intrinsic immune-modulatory properties for the induction of efficient immunity. Like all viral vectored vaccines repeated inoculation via mucosal routes invariably results to immunity against viral vaccine vectors. To obviate immunity against viral vaccine vectors and improve the ability of rNDV vectored vaccines in inducing T cell immunity in murine air way we have directed dendritic cell targeted HIV-1 gag protein (DEC-Gag) vaccine; for the induction of helper CD4 + T cells to a Recombinant Newcastle disease virus expressing codon optimized HIV-1 Gag P55 (rNDV-L-Gag) vaccine. We do so through successive administration of anti-DEC205-gagP24 protein plus polyICLC (DEC-Gag) vaccine and rNDV-L-Gag. First strong gag specific helper CD4 + T cells are induced in mice by selected targeting of anti-DEC205-gagP24 protein vaccine to dendritic cells (DC) in situ together with polyICLC as adjuvant. This targeting helped T cell immunity develop to a subsequent rNDV-L-Gag vaccine and improved both systemic and mucosal gag specific immunity. This sequential DEC-Gag vaccine prime followed by an rNDV-L-gag boost results to improved viral vectored immunization in murine airway, including mobilization of protective CD8 + T cells to a pathogenic virus infection site. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves viral vectored immunization, including mobilization of protective CD8 + T cells to a pathogenic virus infection site such as the murine airway. © 2017 The Authors. Immunity, Inflammation and DiseasePublished by John Wiley & Sons Ltd.
Ge, Jinying; Wang, Xijun; Tao, Lihong; Wen, Zhiyuan; Feng, Na; Yang, Songtao; Xia, Xianzhu; Yang, Chinglai; Chen, Hualan; Bu, Zhigao
Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 109.8 50% egg infective doses (EID50)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 108.3 EID50 completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies. PMID:21632762
Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L
We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation...... of neo or the enhanced green fluorescence protein gene (EGFP). Akv-MLV's with IRES-neo and IRES-EGFP cassettes replicated with titers of about 10(6) infectious units/ml while SL3-3-MLV with IRES-neo gave about 10(3)-fold lower titers. Interestingly, RNA analysis showed a drastic reduction in the amount...
Petukhova, Natalia V; Gasanova, Tatiana V; Stepanova, Liudmila A; Rusova, Oxana A; Potapchuk, Marina V; Korotkov, Alexandr V; Skurat, Eugene V; Tsybalova, Liudmila M; Kiselev, Oleg I; Ivanov, Peter A; Atabekov, Joseph G
A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments proved that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the 2-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/04/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV
Daimei, Guisuibou; Raina, Harpreet Singh; Devi, Pukhrambam Pushpa; Saurav, Gunjan Kumar; Renukadevi, Perumal; Malathi, Varagur Ganesan; Senthilraja, Chinnaiah; Mandal, Bikash; Rajagopal, Raman
The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.
Full Text Available Adenoviruses (Ads are large human-pathogenic double-stranded DNA (dsDNA viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR and linear-circular homologous recombination (LCHR. We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS. We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.
Foy, Brian D; Kobylinski, Kevin C; Chilson Foy, Joy L; Blitvich, Bradley J; Travassos da Rosa, Amelia; Haddow, Andrew D; Lanciotti, Robert S; Tesh, Robert B
Clinical and serologic evidence indicate that 2 American scientists contracted Zika virus infections while working in Senegal in 2008. One of the scientists transmitted this arbovirus to his wife after his return home. Direct contact is implicated as the transmission route, most likely as a sexually transmitted infection.
Duch, Mogens R.; Carrasco, Maria L; Jespersen, Thomas
cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third...
Lommen, S.T.E.; Conijn, C.G.M.; Lemmers, M.E.C.; Pham, K.T.K.; Kock, de M.J.D.
Tulip virus X (TVX) is a Potexvirus causing economic losses in tulip. Potexviruses are generally transmitted by mechanical contact and, indeed, several mechanical transmission pathways for TVX have been identified during tulip bulb production. However, TVX transmission does also seem to occur during
Rasmussen, Lasse Dam; Kristensen, Birgit; Kirkeby, Carsten
, in the south-west of Denmark (close to the German border), were sorted into pools and tested for the presence of Schmallenberg virus RNA by RT-qPCR. From 18 pools of 5 midges from the C. obsoletus group, 2 pools were both found positive in two separate assays, targeting the L- and S- segments of the SBV RNA...
Cuadros, Diego F; Hernandez, Anngie; Torres, Maria F; Torres, Diana M; Branscum, Adam J; Rincon, Diego F
The potato yellow vein disease, caused by the potato yellow vein virus (PYVV), is a limiting potato disease in northern South America. The virus can be transmitted either by the greenhouse whitefly (GWF), Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), or through vegetative propagules, such as infected tubers. Recently, GWF populations have been spotlighted as one of the main drivers of PYVV re-emergence, and consequently, PYVV management has been predominantly directed toward vector control, which is heavily based on insecticide use. However, the drivers of the PYVV outbreaks as well as the contribution of GWF populations on the spread of PYVV among potato crops are still not completely understood. This study aims to assess the role of the GWF as a driver of the PYVV epidemic in the potato-producing areas in Colombia, one of the countries more severely affected by the PYVV epidemic, and whose geography allows the study of the spatial association between the vector and the disease epidemic across a wide altitude range. The geographical clusters where the PYVV epidemic is concentrated, as well as those of farms affected by the GWF were identified using a novel spatial epidemiology approach. The influence of altitude range on the association between PYVV and T. vaporarioum was also assessed. We found a relatively poor spatial association between PYVV epidemic and the presence of the GWF, especially at altitudes above 3,000 m above mean sea level. Furthermore, GWF populations could only explain a small fraction of the extent of the PYVV epidemic in Colombia. Movement of infected seed tubers might be the main mechanism of dispersion, and could be a key driver for the PYVV infection among potato crops. Agricultural policies focused on improving quality of seed tubers and their appropriate distribution could be the most efficient control intervention against PYVV dispersion.
Riezebos-Brilman, Annelies; Regts, Joke; Chen, Margaret; Wilschut, Jan; Daemen, Toos
To enhance the efficacy of a therapeutic immunisition strategy against human papillomavirus-induced cervical cancer we evaluated the adjuvant effect of interleukin-12 (IL12) expressed by a Semliki Forest virus vector (SFV) in mice. Depending on the dose and schedule. SFV-IL12 Stimulated
Conclusions: It is a public health imperative to define the domestic and wild animal reservoirs, amplification hosts, and vector capacity of the genera Aedes, Anopheles, and Mansonia. These variables will define the geographic distribution of Zika virus along with the indicated timing and scale of the environmental public health interventions worldwide.
Withoff, S; Glazenburg, KL; van Veen, ML; Kraak, MMJ; Hospers, GAP; Storkel, S; de Vries, EGE; Wischut, J; Daemen, T
This paper describes the production of recombinant Semliki Forest virus encoding murine or human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the capacity of these vectors to transduce murine and human tumor cells ex vivo. High-titer stocks (up to 3 x 10(9) particles/ml) of
Using reverse genetics technology, many strains of Newcastle disease virus (NDV) have been developed as vectors to express foreign genes for vaccine and gene therapy purposes. The foreign gene is usually inserted into a non-coding region of the NDV genome as an independent transcription unit. Eval...
Weger-Lucarelli, James; Chu, Haiyan; Aliota, Matthew T; Partidos, Charalambos D; Osorio, Jorge E
Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. Outbreaks are associated with high morbidity and create a public health challenge for countries affected. Recent outbreaks have occurred in both Europe and the Americas, suggesting CHIKV may continue to spread. Despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against CHIKV. Therefore, it is critical to develop a vaccine that is both well tolerated and highly protective. In this study, we describe the construction and characterization of a modified Vaccinia virus Ankara (MVA) virus expressing CHIKV E3 and E2 proteins (MVA-CHIK) that protected several mouse models from challenge with CHIKV. In particular, BALB/c mice were completely protected against viremia upon challenge with CHIKV after two doses of MVA-CHIK. Additionally, A129 mice (deficient in IFNα/β) were protected from viremia, footpad swelling, and mortality. While high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. However, passive transfer of MVA-CHIK immune serum to naïve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by MVA-CHIK. Furthermore, depletion of CD4(+), but not CD8(+) T-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of CD4(+) T-cells in the protection afforded by MVA-CHIK. The results presented herein demonstrate the potential of MVA to effectively express CHIKV E3-E2 proteins and generate protective immune responses. Our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against CHIKV, and provides a framework for the development of novel, more effective vaccine strategies to combat CHIKV.
Full Text Available Chikungunya virus (CHIKV is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. Outbreaks are associated with high morbidity and create a public health challenge for countries affected. Recent outbreaks have occurred in both Europe and the Americas, suggesting CHIKV may continue to spread. Despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against CHIKV. Therefore, it is critical to develop a vaccine that is both well tolerated and highly protective.In this study, we describe the construction and characterization of a modified Vaccinia virus Ankara (MVA virus expressing CHIKV E3 and E2 proteins (MVA-CHIK that protected several mouse models from challenge with CHIKV. In particular, BALB/c mice were completely protected against viremia upon challenge with CHIKV after two doses of MVA-CHIK. Additionally, A129 mice (deficient in IFNα/β were protected from viremia, footpad swelling, and mortality. While high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. However, passive transfer of MVA-CHIK immune serum to naïve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by MVA-CHIK. Furthermore, depletion of CD4(+, but not CD8(+ T-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of CD4(+ T-cells in the protection afforded by MVA-CHIK.The results presented herein demonstrate the potential of MVA to effectively express CHIKV E3-E2 proteins and generate protective immune responses. Our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against CHIKV, and provides a framework for the development of novel, more effective vaccine strategies to combat CHIKV.
Hueging, Kathrin; Weller, Romy; Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W R; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas
Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to
Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector.
Liang, Bo; Ngwuta, Joan O; Herbert, Richard; Swerczek, Joanna; Dorward, David W; Amaro-Carambot, Emerito; Mackow, Natalie; Kabatova, Barbora; Lingemann, Matthias; Surman, Sonja; Yang, Lijuan; Chen, Man; Moin, Syed M; Kumar, Azad; McLellan, Jason S; Kwong, Peter D; Graham, Barney S; Schaap-Nutt, Anne; Collins, Peter L; Munir, Shirin
Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high-quality" RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499-9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. Human respiratory syncytial virus (RSV) and
Jacqueline M. Evans
Full Text Available Introduction. Allograft survival can be prolonged by overexpression of cytoprotective genes such as heme oxygenase-1 (HO-1. Modifications in vector design and delivery have provided new opportunities to safely and effectively administer HO-1 into the heart prior to transplantation to improve long-term graft outcome. Methods. HO-1 was delivered to the donor heart using an adeno-associated virus vector (AAV with a pseudotype 6 capsid and vascular endothelial growth factor (VEGF to enhance myocardial tropism and microvascular permeability. Survival of mouse cardiac allografts, fully or partially mismatched at the MHC, was determined with and without cyclosporine A. Intragraft cytokine gene expression was examined by PCR. Results. The use of AAV6 to deliver HO-1 to the donor heart, combined with immunosuppression, prolonged allograft survival by 55.3% when donor and recipient were completely mismatched at the MHC and by 94.6% if partially mismatched. The combination of gene therapy and immunosuppression was more beneficial than treatment with either AAV6-HO-1 or CsA alone. IL-17a, b, e and f were induced in the heart at rejection. Conclusions. Pretreatment of cardiac allografts with AAV6-HO-1 plus cyclosporine A prolonged graft survival. HO-1 gene therapy represents a beneficial adjunct to immunosuppressive therapy in cardiac transplantation.
Full Text Available Dengue fever (DF is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence. Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.
Morin, Cory W.; Comrie, Andrew C.
Climate can strongly influence the population dynamics of disease vectors and is consequently a key component of disease ecology. Future climate change and variability may alter the location and seasonality of many disease vectors, possibly increasing the risk of disease transmission to humans. The mosquito species Culex quinquefasciatus is a concern across the southern United States because of its role as a West Nile virus vector and its affinity for urban environments. Using established relationships between atmospheric variables (temperature and precipitation) and mosquito development, we have created the Dynamic Mosquito Simulation Model (DyMSiM) to simulate Cx. quinquefasciatus population dynamics. The model is driven with climate data and validated against mosquito count data from Pasco County, Florida and Coachella Valley, California. Using 1-week and 2-week filters, mosquito trap data are reproduced well by the model ( P < 0.0001). Dry environments in southern California produce different mosquito population trends than moist locations in Florida. Florida and California mosquito populations are generally temperature-limited in winter. In California, locations are water-limited through much of the year. Using future climate projection data generated by the National Center for Atmospheric Research CCSM3 general circulation model, we applied temperature and precipitation offsets to the climate data at each location to evaluate mosquito population sensitivity to possible future climate conditions. We found that temperature and precipitation shifts act interdependently to cause remarkable changes in modeled mosquito population dynamics. Impacts include a summer population decline from drying in California due to loss of immature mosquito habitats, and in Florida a decrease in late-season mosquito populations due to drier late summer conditions.
Hadaczek, Piotr; Forsayeth, John; Mirek, Hanna; Munson, Keith; Bringas, John; Pivirotto, Phil; McBride, Jodi L; Davidson, Beverly L; Bankiewicz, Krystof S
We used convection-enhanced delivery (CED) to characterize gene delivery mediated by adeno-associated virus type 1 (AAV1) by tracking expression of hrGFP (humanized green fluorescent protein from Renilla reniformis) into the striatum, basal forebrain, and corona radiata of monkey brain. Four cynomolgus monkeys received single infusions into corona radiata, putamen, and caudate. The other group (n = 4) received infusions into basal forebrain. Thirty days after infusion animals were killed and their brains were processed for immunohistochemical evaluation. Volumetric analysis of GFP-positive brain areas was performed. AAV1-hrGFP infusions resulted in approximately 550, 700, and 73 mm(3) coverage after infusion into corona radiata, striatum, and basal forebrain, respectively. Aside from targeted regions, other brain structures also showed GFP signal (internal and external globus pallidus, subthalamic nucleus), supporting the idea that AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant nonneuronal cell population was transduced by AAV1 vector; for example, oligodendrocytes in corona radiata and astrocytes in the striatum. We observed a strong humoral and cell-mediated response against AAV1-hrGFP in transduced monkeys irrespective of the anatomic location of the infusion, as evidenced by induction of circulating anti-AAV1 and anti-hrGFP antibodies, as well as infiltration of CD4(+) lymphocytes and upregulation of MHC-II in regions infused with vector. We conclude that transduction of antigen-presenting cells within the CNS is a likely cause of this response and that caution is warranted when foreign transgenes are used as reporters in gene therapy studies with vectors with broader tropism than AAV2.
Mikkelsen, J G; Lund, Anders Henrik; Duch, M
-impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region....... We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.......Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site...
Mikkelsen, J G; Lund, Anders Henrik; Kristensen, K D
Transduction of primer binding site-impaired Akv murine leukemia virus-based retroviral vectors from the murine packaging cell lines psi-2 and omega E was studied. The efficiency of transduction of the neo marker of all mutated constructs was found to decrease by 5 to 6 orders of magnitude compared...... with that of the wild-type vector. Thirty-two of 60 transduced proviruses analyzed harbored a primer binding site sequence matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pattern of nucleotide differences from the Akv-based vector......, suggesting the involvement of a specific endogenous virus-like sequence in patch repair rescue of the primer binding site mutants. The putative recombination partner RNA was found in virions from psi-2 cells as detected by analysis of glutamine tRNA-initiated cDNA and by sequence analysis of regions...
Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Yang, Hee-Jong; Chwae, Yong-Joon; Kim, Kyongmin; Park, Sun; Shin, Ho-Joon
Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.
Full Text Available We investigated the protective effects of a viral vector based on an Apple latent spherical virus (ALSV harboring a segment of the Bean yellow mosaic virus (BYMV genome against mosaic diseases in pea, broad bean, and eustoma plants caused by BYMV infection. In pea plants pre-inoculated with the ALSV vaccine and challenge inoculated with BYMV expressing green fluorescence protein, BYMV multiplication occurred in inoculated leaves, but was markedly inhibited in the upper leaves. No mosaic symptoms due to BYMV infection were observed in the challenged plants pre-inoculated with the ALSV vaccine. Simultaneous inoculation with the ALSV vaccine and BYMV also prevented mosaic symptoms in broad bean and eustoma plants, and BYMV accumulation was strongly inhibited in the upper leaves of plants treated with the ALSV vaccine. Pea and eustoma plants were pre-inoculated with BYMV followed by inoculation with the ALSV vaccine to investigate the curative effects of the ALSV vaccine. In both plant species, recovery from mosaic symptoms was observed in upper leaves and BYMV accumulation was inhibited in leaves developing post-ALSV vaccination. These results show that ALSV vaccination not only prevents mosaic diseases in pea, broad bean, and eustoma, but that it is also effective in curing these diseases.
O'Donnell, Vivian; Holinka, Lauren G; Gladue, Douglas P; Sanford, Brenton; Krug, Peter W; Lu, Xiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R; Borca, Manuel V
African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs
Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.
Balakrishnan, Balaji; Sen, Dwaipayan; Hareendran, Sangeetha; Roshini, Vaani; David, Sachin; Srivastava, Alok; Jayandharan, Giridhara R
The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12-48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.
Monahan, Paul E; Lothrop, Clinton D; Sun, Junjiang; Hirsch, Matthew L; Kafri, Tal; Kantor, Boris; Sarkar, Rita; Tillson, D Michael; Elia, Joseph R; Samulski, R Jude
Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.
Monahan, Paul E; Lothrop, Clinton D; Sun, Junjiang; Hirsch, Matthew L; Kafri, Tal; Kantor, Boris; Sarkar, Rita; Tillson, D Michael; Elia, Joseph R; Samulski, R Jude
Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration–approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 1013 vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy. PMID:20700109
Davis, Adam S; Federici, Thais; Ray, William C; Boulis, Nicholas M; OʼConnor, Deirdre; Clark, K Reed; Bartlett, Jeffrey S
After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.
Becker, Nathalie; Rimbaud, Loup; Chiroleu, Frédéric; Reynaud, Bernard; Thébaud, Gaël; Lett, Jean-Michel
Of worldwide economic importance, Tomato yellow leaf curl virus (TYLCV, Begomovirus) is responsible for one of the most devastating plant diseases in warm and temperate regions. The DNA begomoviruses (Geminiviridae) are transmitted by the whitefly species complex Bemisia tabaci. Although geminiviruses have long been described as circulative non-propagative viruses, observations such as long persistence of TYLCV in B. tabaci raised the question of their possible replication in the vector. We monitored two major TYLCV strains, Mild (Mld) and Israel (IL), in the invasive B. tabaci Middle East-Asia Minor 1 cryptic species, during and after the viral acquisition, within two timeframes (0–144 hours or 0–20 days). TYLCV DNA was quantified using real-time PCR, and the complementary DNA strand of TYLCV involved in viral replication was specifically quantified using anchored real-time PCR. The DNA of both TYLCV strains accumulated exponentially during acquisition but remained stable after viral acquisition had stopped. Neither replication nor vertical transmission were observed. In conclusion, our quantification of the viral loads and complementary strands of both Mld and IL strains of TYLCV in B. tabaci point to an efficient accumulation and preservation mechanism, rather than to a dynamic equilibrium between replication and degradation. PMID:26625871
Full Text Available Rice stripe mosaic virus (RSMV is a newly discovered species of cytorhabdovirus infecting rice plants that is transmitted by the leafhopper Recilia dorsalis. In this study, the transmission characteristics of RSMV by R. dorsalis were investigated. Under suitable growth conditions for R. dorsalis, the RSMV acquisition rate reached 71.9% in the second-generation population raised on RSMV-infected rice plants. The minimum acquisition and inoculation access periods of R. dorsalis were 3 and 30 min, respectively. The minimum and maximum latent transmission periods of RSMV in R. dorsalis were 6 and 18 d, respectively, and some R. dorsalis intermittently transmitted RSMV at 2–6 d intervals. Our findings revealed that the virus can replicate in the leafhopper body, but is likely not transovarially transmitted to offspring. These transmission characteristics will help guide the formulation of RSMV prevention and control strategies.
Gorch, C; Vagnozzi, A; Duffy, S; Miquet, J; Pacheco, J; Bolondi, A; Draghi, G; Cetra, B; Soni, C; Ronderos, M; Russo, S; Ramírez, V; Lager, I
To establish if BTV was circulating in Argentina, 94 bovines from the Santo Tomé and Ituzaingó Departments of Corrientes Province were sampled every 30-60 days during 14 months. Red blood cells from those animals that showed seroconvertion with a c-ELISA were processed for virus isolation by inoculation in embryonated chicken eggs and cell cultures. Cells with CPE were positive by direct and indirect immunofluorescence with BTV specific reagents. These samples examined by electron microscopy showed virus particles with BTV morphological characteristics. Blood samples and tissue culture supernantants were positive by RT-PCR technique with primers corresponding to the segment 3 of the BTV genome. Haematophagous insects were captured in one farm using light traps and Culicoides insignis Lutz was the predominant species detected. This is the first isolation of BTV in Argentina from northeastern bovines without any disease symptom.
Johnson, J. Erik; Nasar, Farooq; Coleman, John W.; Price, Roger E; Javadian, Ali; Draper, Kenneth; Lee, Margaret; Reilly, Patricia A.; Clarke, David K.; Hendry, R. Michael; Udem, Stephen A.
Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for one day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two ...
O'Brien, Valerie A.; Moore, Amy T.; Young, Ginger R.; Komar, Nicholas; Reisen, William K.; Brown, Charles R.
Determining the effect of an invasive species on enzootic pathogen dynamics is critical for understanding both human epidemics and wildlife epizootics. Theoretical models suggest that when a naive species enters an established host–parasite system, the new host may either reduce (‘dilute’) or increase (‘spillback’) pathogen transmission to native hosts. There are few empirical data to evaluate these possibilities, especially for animal pathogens. Buggy Creek virus (BCRV) is an arthropod-borne...
Kuo, Szu-Cheng; Chen, Ying-Ju; Wang, Yu-Ming; Kuo, Ming-Der; Jinn, Tzyy-Rong; Chen, Wen-Shuo; Chang, Yen-Chung; Tung, Kuo-Lun; Wu, Tzong-Yuan; Lo, Szecheng J
Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion. Copyright © 2011 Elsevier B.V. All rights reserved.
Gu, Hongjing; Li, Tieling; Han, Lina; Zhu, Ping; Zhang, Peirui; Zhang, Shaogeng; Sun, Sujing; Duan, Yueqiang; Xing, Li; Zhao, Zhongpeng; Lai, Chengcai; Wen, Bohai; Wang, Xiliang; Yang, PengHui
Respiratory syncytial virus (RSV) is a major pathogen in infants and the elderly, causing pneumonia and bronchiolitis. Despite decades of research, to date there is still no approved RSV vaccine available. In this study, we developed RSV virus-like particle (VLP) vaccines containing an RSV fusion (F) and/or attachment (G) protein with Newcastle disease virus (NDV) as the platform. The VLPs were expressed in a baculovirus system and purified by sucrose gradient centrifugation. BALB/c mice immunized intranasally (i.n.) with rNDV/RSV/F plus rNDV/RSV/G developed robust humoral, mucosal RSV-specific antibodies and cellular immune responses. Furthermore, rNDV/RSV/F plus rNDV/RSV/G provided better protection than did rNDV/RSV/F or rNDV/RSV/G alone, as shown by an obvious decrease in viral replication together with alleviation of histopathological changes in the lungs of the challenged mice. Our data demonstrate that the intranasal vaccination of combined RSV virus-like particle vaccine candidates has great potential for protection against RSV infection.
Roy, Gourgopal; Sudarshana, Mysore R; Ullman, Diane E; Ding, Shou-Wei; Dandekar, Abhaya M; Falk, Bryce W
ABSTRACT RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3' untranslated region (UTR); and pCTV2 contained CP, p23, and 3' UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.
Trawinski, P. R.; Mackay, D. S.
The objective of this study is to quantify and model spatial dependence in mosquito vector populations and develop predictions for unsampled locations using geostatistics. Mosquito control program trap sites are often located too far apart to detect spatial dependence but the results show that integration of spatial data over time for Cx. pipiens-restuans and according to meteorological conditions for Ae. vexans enables spatial analysis of sparse sample data. This study shows that mosquito abundance is spatially correlated and that spatial dependence differs between Cx. pipiens-restuans and Ae. vexans mosquitoes.
Xiao, Weidong; Chirmule, Narendra; Berta, Scott C.; McCullough, Beth; Gao, Guangping; Wilson, James M.
The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is ...
Shevtsova, Z; Malik, J M I; Michel, U; Bähr, M; Kügler, S
The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.
Ahmed O Hassan
Full Text Available The emergence of H5, H7, and H9 avian influenza virus subtypes in humans reveals their pandemic potential. Although human-to-human transmission has been limited, the genetic reassortment of the avian and human/porcine influenza viruses or mutations in some of the genes resulting in virus replication in the upper respiratory tract of humans could generate novel pandemic influenza viruses. Current vaccines do not provide cross protection against antigenically distinct strains of the H5, H7, and H9 influenza viruses. Therefore, newer vaccine approaches are needed to overcome these potential threats. We developed an egg-independent, adenovirus vector-based, multi-epitope (ME vaccine approach using the relatively conserved immunogenic domains of the H5N1 influenza virus [M2 ectodomain (M2e, hemagglutinin (HA fusion domain (HFD, T-cell epitope of nucleoprotein (TNP. and HA α-helix domain (HαD]. Our ME vaccine induced humoral and cell-mediated immune responses and caused a significant reduction in the viral loads in the lungs of vaccinated mice that were challenged with antigenically distinct H5, H7, or H9 avian influenza viruses. These results suggest that our ME vaccine approach provided broad protection against the avian influenza viruses. Further improvement of this vaccine will lead to a pre-pandemic vaccine that may lower morbidity, hinder transmission, and prevent mortality in a pandemic situation before a strain-matched vaccine becomes available.
Gralton, Jan; McLaws, Mary-Louise; Rawlinson, William D
Previous investigations of fomite transmission have focused on the presence of pathogens on inanimate objects in clinical settings. There has been limited investigation of fomite transmission in non-clinical pediatric settings where there is a high prevalence of respiratory virus infections. Over a 5 week period, this study investigated whether the personal clothing of teachers working in childcare centers was contaminated with viral RNA, and potentially could mediate virus transmission. Matched morning and evening clothing and nasal samples were collected for 313 teacher work days (TWDs). Human rhinoviruses (hRV) RNA were detected from samples using real-time PCR. Human rhinovirus RNA was detected in clothing samples on 16 TWDs and in nasal samples on 32 TWDs. There were no TWDs when teachers provided both positive nasal and clothing samples and only three TWDs when hRV persisted on clothing for the entire day. The detection of hRV RNA was significantly predicted by self-recognition of symptomatic illness by the teacher 2 days prior to detection. These findings suggest that teachers' personal clothing in childcare settings is unlikely to facilitate the transmission of hRV. © 2015 Wiley Periodicals, Inc.
Fernandez-Sainz, Ignacio; Medina, Gisselle N; Ramirez-Medina, Elizabeth; Koster, Marla J; Grubman, Marvin J; de Los Santos, Teresa
A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 107 pfu/animal while a dose of 4×107pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine. Published by Elsevier Inc.
Moore, Amy T.; O'Brien, Valerie A.
Abstract Invasive species can disrupt natural disease dynamics by altering pathogen transmission among native hosts and vectors. The relatively recent occupancy of cliff swallow (Petrochelidon pyrrhonota) nesting colonies in western Nebraska by introduced European house sparrows (Passer domesticus) has led to yearly increases in the prevalence of an endemic arbovirus, Buggy Creek virus (BCRV), in its native swallow bug (Oeciacus vicarius) vector at sites containing both the invasive sparrow host and the native swallow host. At sites without the invasive host, no long-term changes in prevalence have occurred. The percentage of BCRV isolates exhibiting cytopathicity in Vero-cell culture assays increased significantly with year at sites with sparrows but not at swallow-only sites, suggesting that the virus is becoming more virulent to vertebrates in the presence of the invasive host. Increased BCRV prevalence in bug vectors at mixed-species colonies may reflect high virus replication rates in house sparrow hosts, resulting in frequent virus transmission between sparrows and swallow bugs. This case represents a rare empirical example of a pathogen effectively switching to an invasive host, documented in the early phases of the host's arrival in a specialized ecosystem and illustrating how an invasive species can promote long-term changes in host–parasite transmission dynamics. PMID:21923265
Stephen John Fletcher
Full Text Available Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus is the type member of the Tospovirus genus with a broad host range of more than 900 plant species. Interactions between these viruses and their plant hosts and insect vectors via RNA interference pathways are likely a key determinant of pathogenicity. The current investigation, for the first time, compares biogenesis of small RNAs between the plant host and insect vector in the presence or absence of TSWV. Unique viral small interfering RNA (vsiRNA profiles are evident for Arachis hypogaea (peanut and Frankliniella fusca (thrips vector following infection with TSWV. Differences between vsiRNA profiles for these plant and insect species, such as the relative abundance of 21 nt and 22 nt vsiRNAs and locations of alignment hotspots, reflect the diverse siRNA biosynthesis pathways of their respective kingdoms. The presence of unique vsiRNAs in F. fusca samples indicates that vsiRNA generation takes place within the thrips, and not solely through uptake via feeding on vsiRNAs produced in infected A. hypogaea. The study also shows key vsiRNA profile differences for TSWV among plant families, which are evident in the case of A. hypogaea, a legume, and members of Solanaceae (S. lycopersicum and N. benthamiana. Distinctively, overall small RNA biogenesis in A. hypogaea is markedly affected with an absence of the 24 nt small RNAs in TSWV-infected plants, possibly leading to wide-spread molecular and phenotypic perturbations specific to this species. These findings add significant information on the host-virus-vector interaction in terms of RNAi pathways and may lead to better crop and vector specific control strategies.
Fletcher, Stephen J; Shrestha, Anita; Peters, Jonathan R; Carroll, Bernard J; Srinivasan, Rajagopalbabu; Pappu, Hanu R; Mitter, Neena
Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus (TSWV) is the type member of the Tospovirus genus with a broad host range of more than 900 plant species. Interactions between these viruses and their plant hosts and insect vectors via RNAi pathways are likely a key determinant of pathogenicity. The current investigation, for the first time, compares biogenesis of small RNAs between the plant host and insect vector in the presence or absence of TSWV. Unique viral small interfering RNA (vsiRNA) profiles are evident for Arachis hypogaea (peanut) and Frankliniella fusca (thrips vector) following infection with TSWV. Differences between vsiRNA profiles for these plant and insect species, such as the relative abundance of 21 and 22 nt vsiRNAs and locations of alignment hotspots, reflect the diverse siRNA biosynthesis pathways of their respective kingdoms. The presence of unique vsiRNAs in F. fusca samples indicates that vsiRNA generation takes place within the thrips, and not solely through uptake via feeding on vsiRNAs produced in infected A. hypogaea. The study also shows key vsiRNA profile differences for TSWV among plant families, which are evident in the case of A. hypogaea, a legume, and members of Solanaceae (S. lycopersicum and Nicotiana benthamiana). Distinctively, overall small RNA (sRNA) biogenesis in A. hypogaea is markedly affected with an absence of the 24 nt sRNAs in TSWV-infected plants, possibly leading to wide-spread molecular and phenotypic perturbations specific to this species. These findings add significant information on the host-virus-vector interaction in terms of RNAi pathways and may lead to better crop and vector specific control strategies.
Full Text Available Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp. Because of the capacity of Sendai virus (SeV nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.
Pärn, Kalle; Viru, Liane; Lehto, Taavi; Oskolkov, Nikita; Langel, Ülo; Merits, Andres
Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells. PMID:23861978
Full Text Available Aedes aegypti is the main mosquito vector of the four serotypes of dengue virus (DENV. Previous population genetic and vector competence studies have demonstrated substantial genetic structure and major differences in the ability to transmit dengue viruses in Ae. aegypti populations in Mexico.Population genetic studies revealed that the intersection of the Neovolcanic axis (NVA with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. The mosquito populations north and south of the NVA also differed in their vector competence (VC for dengue serotype 2 virus (DENV2. The average VC rate for Ae. aegypti mosquitoes from populations from north of the NVA was 0.55; in contrast the average VC rate for mosquitoes from populations from south of the NVA was 0.20. Most of this variation was attributable to a midgut infection and escape barriers. In Ae. aegypti north of the NVA 21.5% failed to develop midgut infections and 30.3% of those with an infected midgut failed to develop a disseminated infection. In contrast, south of the NVA 45.2% failed to develop midgut infections and 62.8% of those with an infected midgut failed to develop a disseminated infection.Barriers to gene flow in vector populations may also impact the frequency of genes that condition continuous and epidemiologically relevant traits such as vector competence. Further studies are warranted to determine why the NVA is a barrier to gene flow and to determine whether the differences in vector competence seen north and south of the NVA are stable and epidemiologically significant.
Full Text Available Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.
Pärn, Kalle; Viru, Liane; Lehto, Taavi; Oskolkov, Nikita; Langel, Ülo; Merits, Andres
Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.
Full Text Available Eastern equine encephalitis (EEE virus (Togaviridae, Alphavirus is a highly pathogenic mosquito-borne zoonosis that is responsible for occasional outbreaks of severe disease in humans and equines, resulting in high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. have occurred intermittently with no apparent pattern; however, during the last decade we have witnessed recurring annual emergence where EEE virus activity had been historically rare, and expansion into northern New England where the virus had been previously unknown. In the northeastern U.S., EEE virus is maintained in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine (perching birds in freshwater hardwood swamps. However, the identity of key avian species that serve as principal virus reservoir and amplification hosts has not been established. The efficiency with which pathogen transmission occurs within an avian community is largely determined by the relative reservoir competence of each species and by ecological factors that influence contact rates between these avian hosts and mosquito vectors.Contacts between vector mosquitoes and potential avian hosts may be directly quantified by analyzing the blood meal contents of field-collected specimens. We used PCR-based molecular methods and direct sequencing of the mitochondrial cytochrome b gene for profiling of blood meals in Cs. melanura, in an effort to quantify its feeding behavior on specific vertebrate hosts, and to infer epidemiologic implications in four historic EEE virus foci in the northeastern U.S. Avian point count surveys were conducted to determine spatiotemporal host community composition. Of 1,127 blood meals successfully identified to species level, >99% of blood meals were from 65 avian hosts in 27 families and 11 orders, and only seven were from mammalian hosts representing three species. We developed an
Perozo, F; Villegas, P; Estevez, C; Alvarado, I R; Purvis, L B; Williams, S
The development and use of recombinant vaccine vectors for the expression of poultry pathogens proteins is an active research field. The adeno-associated virus (AAV) is a replication-defective virus member of the family Parvoviridae that has been successfully used for gene delivery in humans and other species. In this experiment, an avian adeno-associated virus (AAAV) expressing the infectious bursal disease virus (IBDV) VP2 protein (rAAAV-VP2) was evaluated for protection against IBDV-virulent challenge. Specific pathogen free (SPF) birds were inoculated with rAAAV-VP2 or with a commercial intermediate IBDV vaccine and then challenged with the Edgar strain. IBDV-specific antibody levels were observed in all vaccinated groups; titers were higher for the commercial vaccine group. The live, commercial vaccine induced adequate protection against morbidity and mortality; nevertheless, initial lymphoid depletion and follicular atrophy related to active viral replication was observed as early as day 14 and persisted up to day 28, when birds were challenged. No bursal tissue damage due to rAAAV-VP2 vaccination was observed. Eight-out-of-ten rAAAV-VP2-vaccinated birds survived the challenge and showed no clinical signs. The bursa:body weight ratio and bursa lesion scores in the rAAAV-VP2 group indicated protection against challenge. Therefore, transgenic expression of the VP2 protein after rAAAV-VP2 vaccination induced protective immunity against IBDV challenge in 80% of the birds, without compromising the bursa of Fabricius. The use of rAAAV virions for gene delivery represents a novel approach to poultry vaccination.
Wang, Anping; Wang, Yongjuan; Wu, Shuang; Zuo, Weiyong; Guo, Changming; Hong, Weiming; Zhu, Shanyuan
Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene. After demonstration of the correct expression of AAAV genes, rAAAV-GFP was produced by triple infection of insect cells or triple transfection of HEK293 cells for comparison purpose. Electron microscopy revealed the formation of typical AAAV particles in the insect cells. Western blotting showed the correct assembly of rAAAV particles with a VP protein ratio similar to that of AAAV. Quantitative PCR showed that the insect cell-produced rAAAV yield was almost 25-fold higher than that produced by HEK293 cells. Fluorescent microscopy showed that the insect cell-produced rAAAV could transfer GFP reporter gene into two avian cell types with similar transfer efficiency to that of HEK293 cell-produced rAAAV. These data suggest that insect cell/baculovirus expression system could be used for scalable production of rAAAV, and the viral vector produced could be used as the gene transfer vehicle for avian cells. Copyright © 2016 Elsevier B.V. All rights reserved.
Kotsakiozi, Panayiota; Gloria-Soria, Andrea; Caccone, Adalgisa; Evans, Benjamin; Schama, Renata; Martins, Ademir Jesus; Powell, Jeffrey R
Aedes aegypti, commonly known as "the yellow fever mosquito", is of great medical concern today primarily as the major vector of dengue, chikungunya and Zika viruses, although yellow fever remains a serious health concern in some regions. The history of Ae. aegypti in Brazil is of particular interest because the country was subjected to a well-documented eradication program during 1940s-1950s. After cessation of the campaign, the mosquito quickly re-established in the early 1970s with several dengue outbreaks reported during the last 30 years. Brazil can be considered the country suffering the most from the yellow fever mosquito, given the high number of dengue, chikungunya and Zika cases reported in the country, after having once been declared "free of Ae. aegypti". We used 12 microsatellite markers to infer the genetic structure of Brazilian Ae. aegypti populations, genetic variability, genetic affinities with neighboring geographic areas, and the timing of their arrival and spread. This enabled us to reconstruct their recent history and evaluate whether the reappearance in Brazil was the result of re-invasion from neighboring non-eradicated areas or re-emergence from local refugia surviving the eradication program. Our results indicate a genetic break separating the northern and southern Brazilian Ae. aegypti populations, with further genetic differentiation within each cluster, especially in southern Brazil. Based on our results, re-invasions from non-eradicated regions are the most likely scenario for the reappearance of Ae. aegypti in Brazil. While populations in the northern cluster are likely to have descended from Venezuela populations as early as the 1970s, southern populations seem to have derived more recently from northern Brazilian areas. Possible entry points are also revealed within both southern and northern clusters that could inform strategies to control and monitor this important arbovirus vector.
Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí
Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062
Tahara, Ichiro; Miyake, Koichi; Hanawa, Hideki; Kurai, Toshiyuki; Hirai, Yukihiko; Ishizaki, Masamichi; Uchida, Eiji; Tajiri, Takashi; Shimada, Takashi
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 x 10(11) viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.
Full Text Available Abstract Background Oncolytic herpes simplex virus (HSV vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. Results We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39
Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude
A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kil...
Huszthy, Peter C; Svendsen, Agnete; Wilson, James M; Kotin, Robert M; Lønning, Per Eystein; Bjerkvig, Rolf; Hoover, Frank
The transduction patterns of recombinant adeno-associated virus serotype 1 (AAV1) and serotype 6 (AAV6) vectors were assessed in human glioblastoma multiforme (GBM) cell lines, in human GBM biopsy spheroids, and in tumor xenografts growing in nude rat brains. All the cell lines tested (A172, D37, GaMg, HF66, and U373Mg) were found to be permissive to both AAV1 and AAV6 vectors, and thus displayed a transduction pattern similar to AAV2 vectors. For every cell line tested, the transduction efficiency displayed by AAV2 vectors was better than by isogenic and isopromoter AAV1 vectors. Transduction efficiency was dependent on the viral particle number used, suggesting that the receptors for these vectors are widely distributed in GBM tissues. Interestingly, AAV1, AAV2, and AAV6 vectors were able to infect and transduce the same cells when added simultaneously to monolayer cultures. Infection of human GBM biopsy spheroids with AAV1 and AAV6 vectors resulted in transgene expression both at the surface layers and in the core of the spheroids. Following injection of AAV1 and AAV6 vectors into human GBM biopsy xenografts growing in nude rat brains, reporter gene expression was seen both in the periphery as well as in the central regions of the tumors. When injected into the normal rat brain, both AAV1 and AAV6 vectors were found to transduce several central nervous system (CNS) regions. The presented results suggest a potential therapeutic role for AAV1 and AAV6 vectors in gene therapy for GBM and also for other CNS malignancies.
Carlos H Dumard
Full Text Available Whole inactivated vaccines (WIVs possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.
Remy-Ziller, Christelle; Germain, Claire; Spindler, Anita; Hoffmann, Chantal; Silvestre, Nathalie; Rooke, Ronald; Bonnefoy, Jean-Yves; Préville, Xavier
Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8⁺ T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate.
Raj K. Singh
Full Text Available Zika virus (ZIKV is the most recent intruder that acquired the status of global threat creating panic and frightening situation to public owing to its rapid spread, attaining higher virulence and causing complex clinical manifestations including microcephaly in newborns and Guillain Barré Syndrome. Alike other flaviviruses, the principal mode of ZIKV transmission is by mosquitoes. Advances in research have provided reliable diagnostics for detecting ZIKV infection, while several drug/therapeutic targets and vaccine candidates have been identified recently. Despite these progresses, currently there is neither any effective drug nor any vaccine available against ZIKV. Under such circumstances and to tackle the problem at large, control measures of which mosquito population control need to be strengthened following appropriate mechanical, chemical, biological and genetic control measures. Apart from this, several other known modes of ZIKV transmission which have gained importance in recent past such as intrauterine, sexual intercourse, and blood-borne spread need to be checked and kept under control by adopting appropriate precautions and utmost care during sexual intercourse, blood transfusion and organ transplantation. The virus inactivation by pasteurization, detergents, chemicals, and filtration can effectively reduce viral load in plasma-derived medicinal products. Added to this, strengthening of the surveillance and monitoring of ZIKV as well as avoiding travel to Zika infected areas would aid in keeping viral infection under check. Here, we discuss the salient advances in the prevention and control strategies to combat ZIKV with a focus on highlighting various intervention approaches against the vector mosquitoes of this viral pathogen along with presenting an overview regarding human intervention measures to counter other modes of ZIKV transmission and spread. Additionally, owing to the success of vaccines for a number of infections
Streck, Christian J; Zhang, Youbin; Zhou, Junfang; Ng, Catherine; Nathwani, Amit C; Davidoff, Andrew M
The purpose of this study was to evaluate the ability of adeno-associated virus (AAV) vector-mediated delivery of pigment epithelium-derived factor (PEDF) to inhibit neuroblastoma (NB) xenograft growth. Pigment epithelium-derived factor was chosen for this study because, in addition to being a potent inhibitor of angiogenesis, it is capable of inducing neuronal differentiation. Cohorts of mice received either recombinant AAV encoding human PEDF (rAAV-hPEDF) at a range of doses or control vector via tail vein. Subsequent hPEDF expression was measured by enzyme-linked immunoassay. After 6 weeks, the mice were given human NB cells by retroperitoneal injection and then killed 5 weeks