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Alexander G Pletnev - One of the best experts on this subject based on the ideXlab platform.
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Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus.
mBio, 2019Co-Authors: Konstantin A. Tsetsarkin, Olga A. Maximova, Guangping Liu, Heather Kenney, Natalya L. Teterina, Alexander G PletnevAbstract:Tick-borne encephalitis Virus (TBEV), a member of the genus FlaviVirus, is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, attempts to develop of a live attenuated Virus (LAV) vaccine against TBEV with acceptable safety and immunogenicity characteristics have not been successful. To overcome this impasse, we generated a chimeric TBEV that was highly immunogenic in nonhuman primates (NHPs). The chimeric Virus contains the prM/E genes of TBEV, which are expressed in the genetic background of an antigenically closely related, but less pathogenic member of the TBEV complex-Langat Virus (LGTV), strain T-1674. The neurovirulence of this chimeric Virus was subsequently controlled by robust targeting of the viral genome with multiple copies of central nervous system-enriched microRNAs (miRNAs). This miRNA-targeted T/1674-mirV2 Virus was highly stable in Vero cells and was not pathogenic in various mouse models of infection or in NHPs. Importantly, in NHPs, a single dose of the T/1674-mirV2 Virus induced TBEV-specific neutralizing antibody (NA) levels comparable to those seen with a three-dose regimen of an inactivated TBEV vaccine, currently available in Europe. Moreover, our vaccine candidate provided complete protection against a stringent wild-type TBEV challenge in mice and against challenge with a parental (not miRNA-targeted) chimeric TBEV/LGTV in NHPs. Thus, this highly attenuated and immunogenic T/1674-mirV2 Virus is a promising LAV vaccine candidate against TBEV and warrants further preclinical evaluation of its neurovirulence in NHPs prior to entering clinical trials in humans.IMPORTANCE Tick-borne encephalitis Virus (TBEV) is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, efforts to develop of TBEV live attenuated Virus (LAV) vaccines with acceptable safety and immunogenicity characteristics have not been successful. Here we report the development and evaluation of a highly attenuated and immunogenic microRNA-targeted TBEV LAV.
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Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus
American Society for Microbiology, 2019Co-Authors: Konstantin A. Tsetsarkin, Olga A. Maximova, Guangping Liu, Heather Kenney, Natalya L. Teterina, Alexander G PletnevAbstract:Tick-borne encephalitis Virus (TBEV) is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, efforts to develop of TBEV live attenuated Virus (LAV) vaccines with acceptable safety and immunogenicity characteristics have not been successful. Here we report the development and evaluation of a highly attenuated and immunogenic microRNA-targeted TBEV LAV.Tick-borne encephalitis Virus (TBEV), a member of the genus FlaviVirus, is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, attempts to develop of a live attenuated Virus (LAV) vaccine against TBEV with acceptable safety and immunogenicity characteristics have not been successful. To overcome this impasse, we generated a chimeric TBEV that was highly immunogenic in nonhuman primates (NHPs). The chimeric Virus contains the prM/E genes of TBEV, which are expressed in the genetic background of an antigenically closely related, but less pathogenic member of the TBEV complex—Langat Virus (LGTV), strain T-1674. The neurovirulence of this chimeric Virus was subsequently controlled by robust targeting of the viral genome with multiple copies of central nervous system-enriched microRNAs (miRNAs). This miRNA-targeted T/1674-mirV2 Virus was highly stable in Vero cells and was not pathogenic in various mouse models of infection or in NHPs. Importantly, in NHPs, a single dose of the T/1674-mirV2 Virus induced TBEV-specific neutralizing antibody (NA) levels comparable to those seen with a three-dose regimen of an inactivated TBEV vaccine, currently available in Europe. Moreover, our vaccine candidate provided complete protection against a stringent wild-type TBEV challenge in mice and against challenge with a parental (not miRNA-targeted) chimeric TBEV/LGTV in NHPs. Thus, this highly attenuated and immunogenic T/1674-mirV2 Virus is a promising LAV vaccine candidate against TBEV and warrants further preclinical evaluation of its neurovirulence in NHPs prior to entering clinical trials in humans
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FlaviVirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control.
Mbio, 2017Co-Authors: Jeffrey M Grabowski, Luwanika Mlera, Danielle K Offerdahl, Alexander G Pletnev, Konstantin A. Tsetsarkin, Rebecca Rosenke, Dana P Scott, Tom G. Schwan, Dan Long, Marshall E BloomAbstract:Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviViruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan Virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat Virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of Virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of Virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flaviVirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan Virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat Virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.
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Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell–Mediated Viral Clearance
Journal of Immunology, 2016Co-Authors: Daniela Michlmayr, Susana V. Bardina, Carlos A Rodriguez, Alexander G PletnevAbstract:Tick-borne encephalitis Virus (TBEV) is a vector-transmitted flaviVirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing worldwide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flaviVirus Langat Virus (LGTV). Ccr5-deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV-infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr5−/− mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr5−/− mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with isotype control–treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS.
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kissing loop interaction between 5 and 3 ends of tick borne Langat Virus genome bridges the gap between mosquito and tick borne flaviViruses in mechanisms of viral rna cyclization applications for Virus attenuation and vaccine development
Nucleic Acids Research, 2016Co-Authors: Konstantin A. Tsetsarkin, Kui Shen, Guangping Liu, Alexander G PletnevAbstract:Insertion of microRNA target sequences into the flaviVirus genome results in selective tissue-specific attenuation and host-range restriction of live attenuated vaccine Viruses. However, previous strategies for miRNA-targeting did not incorporate a mechanism to prevent target elimination under miRNA-mediated selective pressure, restricting their use in vaccine development. To overcome this limitation, we developed a new approach for miRNA-targeting of tick-borne flaviVirus (Langat Virus, LGTV) in the duplicated capsid gene region (DCGR). Genetic stability of Viruses with DCGR was ensured by the presence of multiple cis-acting elements within the N-terminal capsid coding region, including the stem-loop structure (5'SL6) at the 3' end of the promoter. We found that the 5'SL6 functions as a structural scaffold for the conserved hexanucleotide motif at its tip and engages in a complementary interaction with the region present in the 3' NCR to enhance viral RNA replication. The resulting kissing-loop interaction, common in tick-borne flaviViruses, supports a single pair of cyclization elements (CYC) and functions as a homolog of the second pair of CYC that is present in the majority of mosquito-borne flaviViruses. Placing miRNA targets into the DCGR results in superior attenuation of LGTV in the CNS and does not interfere with development of protective immunity in immunized mice.
Tetsuya Tanaka - One of the best experts on this subject based on the ideXlab platform.
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An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat Virus infection of a tick cell line.
Acta tropica, 2020Co-Authors: Emmanuel Pacia Hernandez, Kentaro Yoshii, Melbourne Rio Talactac, Rodel Jonathan Santos Vitor, Tetsuya TanakaAbstract:Ticks are important vectors of diseases affecting both humans and animals. To be an efficient vector, ticks have to survive infection by pathogens such as the Langat Virus (LGTV). One method utilized by ticks is their complex antioxidant mechanism. Included in the vast antioxidant processes are several enzymes involved in redox homeostasis. The ubiquitous glutathione S-transferases (GSTs) belong to the antioxidant family of enzymes. In this study, we evaluated the role of a GST during LGTV infection. ISE6 cells were infected with LGTV with a multiplicity of infection (MOI) of 0.01 and observed daily. The infection success was monitored via indirect immunofluorescent antibody test (IFAT) for LGTV for up to 4 days. The gene expression of IsGST1 was determined by real-time polymerase chain reaction (PCR) using IsGST1 gene-specific primers. Knockdown of the IsGST1 gene with subsequent LGTV infection was also performed. Afterward, ISE6 cell mortality and viability were checked daily until the fourth day. The Virus titer from supernatants of IsGST1-knockdown cells was quantified using a focus-formation assay. IFAT data showed that LGTV infects ISE6 cells in a time-dependent manner with increasing infection from day 0 to day 4. The IsGST1 genes showed an increasing expression until day 2 of infection, while decreased expression was observed from day 3 to day 4 post-infection. Knockdown of the IsGST1 resulted in increased mortality on the third day of infection, while the cell viability was also negatively affected by the knockdown of the IsGST1 genes from day 0 to day 4 post-infection. Knockdown of the IsGST1 genes also resulted in a decreased viral titer from the supernatants of the ISE6 cells infected with LGTV. Based on the results, GSTs are possibly utilized both by cells and the Virus for mutual survival and proliferation.
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A Peroxiredoxin From the Haemaphysalis longicornis Tick Affects Langat Virus Replication in a Hamster Cell Line.
Frontiers in cellular and infection microbiology, 2020Co-Authors: Kodai Kusakisako, Kentaro Yoshii, Emmanuel Pacia Hernandez, Melbourne Rio Talactac, Haruki Morokuma, Tetsuya TanakaAbstract:Ticks are hematophagous arthropods, and their blood feeding on vertebrate hosts is essential for their development. The vertebrate blood contains high levels of free iron that can react with oxygen in ticks, resulting in the production of hydrogen peroxide (H2O2), one of the reactive oxygen species. Peroxiredoxins (Prxs), H2O2-scavenging enzymes, take on an important role in the ticks' oxidative stress coping mechanism. Ticks also transmit several disease-causing pathogens, including tick-borne encephalitis Virus (TBEV), in animals and humans. Therefore, the control of ticks and tick-borne pathogens is a key issue that needs to be addressed. Infection with an arthropod-borne flaviVirus is known to induce oxidative stress in insect cells. We hypothesize that vector-derived Prxs could have an effect on the infection and/or replication of flaviViruses in the hosts, since ticks Prxs are possibly transmitted from ticks to their hosts. In this study, we established stable strains of baby hamster kidney (BHK) cells expressing two types of H2O2-scavenging Prxs from the hard tick Haemaphysalis longicornis (BHK-HlPrx and BHK-HlPrx2 cells). Although the infection of TBEV surrogate Langat Virus (LGTV) did not induce H2O2 production in normal BHK cells, the mortality rate and the Virus titer of LGTV infected BHK-HlPrx cells increased. In addition, HlPrx proteins in BHK cells can facilitate LGTV replication in cells, while HlPrx2 proteins in BHK cells cannot. The results also demonstrated that this facilitation of LGTV replication by the 1-Cys Prx in the BHK cells is not by scavenging H2O2 but by an unknown mechanism. In order to understand this mechanism, more studies using tick-derived cells and ticks are necessary.
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Synchronous Langat Virus Infection of Haemaphysalis longicornis Using Anal Pore Microinjection
Viruses, 2017Co-Authors: Melbourne Rio Talactac, Kentaro Yoshii, Kodai Kusakisako, Remil Linggatong Galay, Emmanuel Pacia Hernandez, Masami Mochizuki, Kozo Fujisaki, Tetsuya TanakaAbstract:The tick-borne encephalitis Virus (TBEV) serocomplex of flaviViruses consists of arboViruses that cause important diseases in animals and humans. The transmission of this group of Viruses is commonly associated with tick species such as Ixodes spp., Dermacentor spp., and Hyalomma spp. In the case of Haemaphysalis longicornis, the detection and isolation of flaviViruses have been previously reported. However, studies showing survival dynamics of any tick-borne flaviVirus in H. longicornis are still lacking. In this study, an anal pore microinjection method was used to infect adult H. longicornis with Langat Virus (LGTV), a naturally attenuated member of the TBEV serocomplex. LGTV detection in ticks was done by real-time PCR, Virus isolation, and indirect immunofluorescent antibody test. The maximum viral titer was recorded at 28 days post-inoculation, and midgut cells were shown to be the primary replication site. The tick can also harbor the Virus for at least 120 days and can successfully transmit LGTV to susceptible mice as confirmed by detection of LGTV antibodies. However, no transovarial transmission was observed from the egg and larval samples. Taken together, our results highly suggest that anal pore microinjection can be an effective method in infecting adult H. longicornis, which can greatly assist in our efforts to study tick and Virus interactions.
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Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis.
Developmental and comparative immunology, 2016Co-Authors: Melbourne Rio Talactac, Kentaro Yoshii, Kodai Kusakisako, Remil Linggatong Galay, Emmanuel Pacia Hernandez, Masami Mochizuki, Kozo Fujisaki, Hiroki Maeda, Yurika Yada, Tetsuya TanakaAbstract:Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat Virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenoVirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the Virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.
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Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis Virus surrogate Langat Virus
Parasites & Vectors, 2016Co-Authors: Melbourne Rio Talactac, Kentaro Yoshii, Kodai Kusakisako, Remil Linggatong Galay, Emmanuel Pacia Hernandez, Naotoshi Tsuji, Kozo Fujisaki, Hiroki Maeda, Tetsuya Tanaka, Masami MochizukiAbstract:Background Longicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat Virus (LGTV), a tick-borne flaviVirus.
Melbourne Rio Talactac - One of the best experts on this subject based on the ideXlab platform.
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An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat Virus infection of a tick cell line.
Acta tropica, 2020Co-Authors: Emmanuel Pacia Hernandez, Kentaro Yoshii, Melbourne Rio Talactac, Rodel Jonathan Santos Vitor, Tetsuya TanakaAbstract:Ticks are important vectors of diseases affecting both humans and animals. To be an efficient vector, ticks have to survive infection by pathogens such as the Langat Virus (LGTV). One method utilized by ticks is their complex antioxidant mechanism. Included in the vast antioxidant processes are several enzymes involved in redox homeostasis. The ubiquitous glutathione S-transferases (GSTs) belong to the antioxidant family of enzymes. In this study, we evaluated the role of a GST during LGTV infection. ISE6 cells were infected with LGTV with a multiplicity of infection (MOI) of 0.01 and observed daily. The infection success was monitored via indirect immunofluorescent antibody test (IFAT) for LGTV for up to 4 days. The gene expression of IsGST1 was determined by real-time polymerase chain reaction (PCR) using IsGST1 gene-specific primers. Knockdown of the IsGST1 gene with subsequent LGTV infection was also performed. Afterward, ISE6 cell mortality and viability were checked daily until the fourth day. The Virus titer from supernatants of IsGST1-knockdown cells was quantified using a focus-formation assay. IFAT data showed that LGTV infects ISE6 cells in a time-dependent manner with increasing infection from day 0 to day 4. The IsGST1 genes showed an increasing expression until day 2 of infection, while decreased expression was observed from day 3 to day 4 post-infection. Knockdown of the IsGST1 resulted in increased mortality on the third day of infection, while the cell viability was also negatively affected by the knockdown of the IsGST1 genes from day 0 to day 4 post-infection. Knockdown of the IsGST1 genes also resulted in a decreased viral titer from the supernatants of the ISE6 cells infected with LGTV. Based on the results, GSTs are possibly utilized both by cells and the Virus for mutual survival and proliferation.
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A Peroxiredoxin From the Haemaphysalis longicornis Tick Affects Langat Virus Replication in a Hamster Cell Line.
Frontiers in cellular and infection microbiology, 2020Co-Authors: Kodai Kusakisako, Kentaro Yoshii, Emmanuel Pacia Hernandez, Melbourne Rio Talactac, Haruki Morokuma, Tetsuya TanakaAbstract:Ticks are hematophagous arthropods, and their blood feeding on vertebrate hosts is essential for their development. The vertebrate blood contains high levels of free iron that can react with oxygen in ticks, resulting in the production of hydrogen peroxide (H2O2), one of the reactive oxygen species. Peroxiredoxins (Prxs), H2O2-scavenging enzymes, take on an important role in the ticks' oxidative stress coping mechanism. Ticks also transmit several disease-causing pathogens, including tick-borne encephalitis Virus (TBEV), in animals and humans. Therefore, the control of ticks and tick-borne pathogens is a key issue that needs to be addressed. Infection with an arthropod-borne flaviVirus is known to induce oxidative stress in insect cells. We hypothesize that vector-derived Prxs could have an effect on the infection and/or replication of flaviViruses in the hosts, since ticks Prxs are possibly transmitted from ticks to their hosts. In this study, we established stable strains of baby hamster kidney (BHK) cells expressing two types of H2O2-scavenging Prxs from the hard tick Haemaphysalis longicornis (BHK-HlPrx and BHK-HlPrx2 cells). Although the infection of TBEV surrogate Langat Virus (LGTV) did not induce H2O2 production in normal BHK cells, the mortality rate and the Virus titer of LGTV infected BHK-HlPrx cells increased. In addition, HlPrx proteins in BHK cells can facilitate LGTV replication in cells, while HlPrx2 proteins in BHK cells cannot. The results also demonstrated that this facilitation of LGTV replication by the 1-Cys Prx in the BHK cells is not by scavenging H2O2 but by an unknown mechanism. In order to understand this mechanism, more studies using tick-derived cells and ticks are necessary.
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Synchronous Langat Virus Infection of Haemaphysalis longicornis Using Anal Pore Microinjection
Viruses, 2017Co-Authors: Melbourne Rio Talactac, Kentaro Yoshii, Kodai Kusakisako, Remil Linggatong Galay, Emmanuel Pacia Hernandez, Masami Mochizuki, Kozo Fujisaki, Tetsuya TanakaAbstract:The tick-borne encephalitis Virus (TBEV) serocomplex of flaviViruses consists of arboViruses that cause important diseases in animals and humans. The transmission of this group of Viruses is commonly associated with tick species such as Ixodes spp., Dermacentor spp., and Hyalomma spp. In the case of Haemaphysalis longicornis, the detection and isolation of flaviViruses have been previously reported. However, studies showing survival dynamics of any tick-borne flaviVirus in H. longicornis are still lacking. In this study, an anal pore microinjection method was used to infect adult H. longicornis with Langat Virus (LGTV), a naturally attenuated member of the TBEV serocomplex. LGTV detection in ticks was done by real-time PCR, Virus isolation, and indirect immunofluorescent antibody test. The maximum viral titer was recorded at 28 days post-inoculation, and midgut cells were shown to be the primary replication site. The tick can also harbor the Virus for at least 120 days and can successfully transmit LGTV to susceptible mice as confirmed by detection of LGTV antibodies. However, no transovarial transmission was observed from the egg and larval samples. Taken together, our results highly suggest that anal pore microinjection can be an effective method in infecting adult H. longicornis, which can greatly assist in our efforts to study tick and Virus interactions.
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Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis.
Developmental and comparative immunology, 2016Co-Authors: Melbourne Rio Talactac, Kentaro Yoshii, Kodai Kusakisako, Remil Linggatong Galay, Emmanuel Pacia Hernandez, Masami Mochizuki, Kozo Fujisaki, Hiroki Maeda, Yurika Yada, Tetsuya TanakaAbstract:Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat Virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenoVirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the Virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.
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Virucidal activity of Haemaphysalis longicornis longicin P4 peptide against tick-borne encephalitis Virus surrogate Langat Virus
Parasites & Vectors, 2016Co-Authors: Melbourne Rio Talactac, Kentaro Yoshii, Kodai Kusakisako, Remil Linggatong Galay, Emmanuel Pacia Hernandez, Naotoshi Tsuji, Kozo Fujisaki, Hiroki Maeda, Tetsuya Tanaka, Masami MochizukiAbstract:Background Longicin is a defensin-like peptide, identified from the midgut epithelium of hard tick Haemaphysalis longicornis. Several studies have already shown the antimicrobial and parasiticidal activities of longicin peptide and one of its synthetic partial analogs, longicin P4. In this study, longicin peptides were tested for potential antiviral activity against Langat Virus (LGTV), a tick-borne flaviVirus.
Kathryn A. Hanley - One of the best experts on this subject based on the ideXlab platform.
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Replacement of the 3′ untranslated variable region of mosquito-borne dengue Virus with that of tick-borne Langat Virus does not alter vector specificity
Journal of General Virology, 2011Co-Authors: Ebenezer Tumban, Dana N. Mitzel, Nyree E. Maes, Christopher T. Hanson, Stephen S. Whitehead, Kathryn A. HanleyAbstract:The four major flaviVirus clades are transmitted by mosquitoes, ticks, directly between vertebrates or directly between arthropods, respectively, but the molecular determinants of mode of transmission in flaviViruses are unknown. To assess the role of the UTRs in transmission, we generated chimeric genomes in which the 5′ UTR, capsid and/or 3′ UTR of mosquito-borne dengue Virus serotype 4 (rDENV-4) were replaced, separately or in combination, with those of tick-borne Langat Virus (rLGTV). None of the chimeric genomes yielded detectable Virus following transfection. Replacement of the variable region (VR) in the rDENV-4 3′ UTR with that of rLGTV generated Virus rDENV-4-rLGTswapVR, which showed lower replication than its wild-type parents in mammalian but not mosquito cells in culture and was able to infect mosquitoes in vivo. Neither rDENV-4 nor rDENV-4-rLGTswapVR could infect larval Ixodes scapularis ticks immersed in Virus, while rLGTV was highly infectious via this route.
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Replacement of the 3' untranslated variable region of mosquito-borne dengue Virus with that of tick-borne Langat Virus does not alter vector specificity.
The Journal of general virology, 2011Co-Authors: Ebenezer Tumban, Dana N. Mitzel, Nyree E. Maes, Christopher T. Hanson, Stephen S. Whitehead, Kathryn A. HanleyAbstract:The four major flaviVirus clades are transmitted by mosquitoes, ticks, directly between vertebrates or directly between arthropods, respectively, but the molecular determinants of mode of transmission in flaviViruses are unknown. To assess the role of the UTRs in transmission, we generated chimeric genomes in which the 5' UTR, capsid and/or 3' UTR of mosquito-borne dengue Virus serotype 4 (rDENV-4) were replaced, separately or in combination, with those of tick-borne Langat Virus (rLGTV). None of the chimeric genomes yielded detectable Virus following transfection. Replacement of the variable region (VR) in the rDENV-4 3' UTR with that of rLGTV generated Virus rDENV-4-rLGTswapVR, which showed lower replication than its wild-type parents in mammalian but not mosquito cells in culture and was able to infect mosquitoes in vivo. Neither rDENV-4 nor rDENV-4-rLGTswapVR could infect larval Ixodes scapularis ticks immersed in Virus, while rLGTV was highly infectious via this route.
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Secondary structure of dengue Virus type 4 3' untranslated region: impact of deletion and substitution mutations.
Journal of General Virology, 2006Co-Authors: Tammy A. Romero, Ebenezer Tumban, Jeongwon Jun, William B. Lott, Kathryn A. HanleyAbstract:Several studies have generated computer-based predictions of secondary structure of the 3' untranslated region (UTR) of Dengue Virus (DEN); however, experimental verification of the formation of these structures in vitro is lacking. This study assessed the congruence of Mfold predictions of secondary structure of the core region of the DEN type 4 3' UTR with nuclease maps of this region. Maps and predictions were largely consistent. Maps supported the existence of previously predicted pseudoknots and identified putative regions of dynamic folding. Additionally, this study investigated previously identified conserved elements in the flaviVirus 3' UTR that differ among Viruses with different modes of transmission. Specific regions of mosquito-borne DEN type 4 were either deleted or replaced with homologous sequences from tick-borne Langat Virus. All of these mutations caused substantial distortion of secondary structure, yet Viruses carrying these mutations were viable.
Marshall E Bloom - One of the best experts on this subject based on the ideXlab platform.
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perk mediated unfolded protein response signaling restricts replication of the tick borne flaviVirus Langat Virus
Viruses, 2020Co-Authors: Tyler G Lewy, Luwanika Mlera, Danielle K Offerdahl, Jeffrey M Grabowski, Eliza M Kellman, Abhilash I Chiramel, Marshall E BloomAbstract:The unfolded protein response (UPR) maintains protein-folding homeostasis in the endoplasmic reticulum (ER) and has been implicated as both beneficial and detrimental to flaviVirus infection. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a sensor of the UPR, is commonly associated with antiviral effects during mosquito-borne flaviVirus (MBFV) infection, but its relation to tick-borne flaviVirus (TBFV) infection remains largely unexplored. In this study, we identified changes in UPR and autophagic activity during Langat Virus (LGTV) infection. LGTV robustly activated UPR and altered autophagic flux. Knockdown of endogenous PERK in human cells resulted in increased LGTV replication, but not that of closely related Powassan Virus (POWV). Finally, on examining changes in protein levels of components associated with UPR and autophagy in the absence of PERK, we could show that LGTV-infected cells induced UPR but did not lead to expression of C/EBP homologous protein (CHOP), an important downstream transcription factor of multiple stress pathways. From these data, we hypothesize that LGTV can antagonize other kinases that target eukaryotic initiation factor 2α (eIF2α), but not PERK, implicating PERK as a potential mediator of intrinsic immunity. This effect was not apparent for POWV, a more pathogenic TBFV, suggesting it may be better equipped to mitigate the antiviral effects of PERK.
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FlaviVirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control.
Mbio, 2017Co-Authors: Jeffrey M Grabowski, Luwanika Mlera, Danielle K Offerdahl, Alexander G Pletnev, Konstantin A. Tsetsarkin, Rebecca Rosenke, Dana P Scott, Tom G. Schwan, Dan Long, Marshall E BloomAbstract:Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviViruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan Virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat Virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of Virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of Virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flaviVirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan Virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat Virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.
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Analysis of the Langat Virus Genome in Persistent Infection of an Ixodes scapularis Cell Line.
Viruses, 2016Co-Authors: Luwanika Mlera, Danielle K Offerdahl, Wessam Melik, Stephen F Porcella, Eric Dahlstrom, Marshall E BloomAbstract:Tick-borne flaviViruses (TBFVs) cause a broad spectrum of disease manifestations ranging from asymptomatic to mild febrile illness and life threatening encephalitis. These single-stranded positive-sense (ss(+)) RNA Viruses are naturally maintained in a persistent infection of ixodid ticks and small-medium sized mammals. The development of cell lines from the ixodid ticks has provided a valuable surrogate system for studying the biology of TBFVs in vitro. When we infected ISE6 cells, an Ixodes scapularis embryonic cell line, with Langat Virus (LGTV) we observed that the infection proceeded directly into persistence without any cytopathic effect. Analysis of the viral genome at selected time points showed that no defective genomes were generated during LGTV persistence by 10 weeks of cell passage. This was in contrast to LGTV persistence in 293T cells in which defective viral genomes are detectable by five weeks of serial cell passage. We identified two synonymous nucleotide changes i.e., 1893A→C (29% of 5978 reads at 12 h post infection (hpi)) and 2284T→A (34% of 4191 reads at 12 hpi) in the region encoding for the viral protein E. These results suggested that the mechanisms supporting LGTV persistence are different between tick and mammalian cells.
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development of a model system for tick borne flaviVirus persistence in hek 293t cells
Mbio, 2015Co-Authors: Luwanika Mlera, Danielle K Offerdahl, Wessam Melik, Stephen F Porcella, Craig Martens, Marshall E BloomAbstract:ABSTRACT We devised a model system to study persistent infection by the tick-borne flaviVirus Langat Virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. IMPORTANCE Tick-borne flaviViruses are significant infectious agents that cause serious disease and death in humans worldwide. Infections are characterized by severe neurological symptoms, such as meningitis and encephalitis. A high percentage of people who get infected and recuperate from the acute phase of infection continue to suffer from chronic debilitating neurological sequelae, most likely as a result of nervous tissue damage, viral persistence, or both. However, little is known about mechanisms of viral persistence. Therefore, we undertook studies to investigate the persistence of Langat Virus, a member of the tick-borne flaviVirus group, in a mammalian cell line. Using next-generation sequencing, we determined that defective viral genomes do not play a role in the initiation of persistence, but their occurrence seems to be nonstochastic and could play a role in the maintenance of viral persistence via the expression of a novel envelope-NS1 fusion protein.
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identification of genetic determinants of a tick borne flaviVirus associated with host specific adaptation and pathogenicity
Virology, 2008Co-Authors: Dana N. Mitzel, Sonja M. Best, Max F Masnick, James B Wolfinbarger, Stephen F Porcella, Marshall E BloomAbstract:Tick-borne flaviViruses are maintained in nature in an enzootic cycle involving a tick vector and a vertebrate host. Thus, the Virus replicates in two disparate hosts, each providing selective pressures that can influence Virus replication and pathogenicity. To identify viral determinants associated with replication in the individual hosts, plaque purified Langat Virus (TP21pp) was adapted to growth in mouse or tick cell lines to generate two Virus variants, MNBp20 and ISEp20, respectively. Virus adaptation to mouse cells resulted in four amino acid changes in MNBp20 relative to TP21pp, occurring in E, NS4A and NS4B. A comparison between TP21pp and ISEp20 revealed three amino acid modifications in M, NS3 and NS4A of ISEp20. ISEp20, but not MNBp20, was attenuated following intraperitoneal inoculation of mice. Following isolation from mice brains, additional mutations reproducibly emerged in E and NS3 of ISEp20 that were possibly compensatory for the initial adaptation to tick cells. Thus, our data implicate a role for E, M, NS3, NS4A and NS4B in host adaptation and pathogenicity of tick-borne flaviViruses.
