The Experts below are selected from a list of 378 Experts worldwide ranked by ideXlab platform
Alexander G Pletnev - One of the best experts on this subject based on the ideXlab platform.
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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, Dana P. Scott, Dan Long, Rebecca Rosenke, Tom G. Schwan, 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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Concurrent micro-RNA mediated silencing of Tick-Borne Flavivirus replication in Tick vector and in the brain of vertebrate host
Scientific Reports, 2016Co-Authors: Konstantin A. Tsetsarkin, Heather Kenney, Meghan Hermance, Saravanan Thangamani, Alexander G PletnevAbstract:Tick-Borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-Borne viruses, whose impact can be restrained via mosquito population control programs, for Tick-Borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable Tick-vertebrate host transmission cycle in nature. To restrict Tick-Borne Flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for Tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in Tick-derived cells. LGTV expressing combinations of target sequences for Tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in Tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus Ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live Tick-Borne virus vaccine candidates.
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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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microrna based control of Tick Borne Flavivirus neuropathogenesis challenges and perspectives
Antiviral Research, 2016Co-Authors: Natalya L Teterina, Heather Kenney, Olga A Maximova, Alexander G PletnevAbstract:Abstract In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenicity of both DNA and RNA viruses. Previously, we reported the successful application of this strategy to control the neurovirulent phenotype of a model chimeric Tick-Borne encephalitis/dengue type 4 virus (TBEV/DEN4), containing the structural protein genes of a highly virulent TBEV in the genetic backbone of non-neuroinvasive DEN4 virus. In the present study, we investigated the suitability of this approach for the attenuation of the more neurovirulent chimeric virus (TBEV/LGTV), which is based on the genetic backbone of the naturally attenuated member of the TBEV serocomplex, a Langat virus (LGTV). Unlike the TBEV/DEN4, the TBEV/LGTV virus retained the ability of its parental viruses to spread from the peripheral site of inoculation to the CNS. We evaluated ten potential sites in the 3′NCR of the TBEV/LGTV genome for placement of microRNA (miRNA) targets and found that the TBEV/LGTV genome is more restrictive for such genetic manipulations compared to TBEV/DEN4. In addition, unlike TBEV/DEN4 virus, the introduction of multiple miRNA targets into either the 3′NCR or ORF of the TBEV/LGTV genome had only a modest effect on virus attenuation in the developing CNS of highly permissive newborn mice. However, simultaneous miRNA-targeting in the ORF and 3′NCR had synergistic effect on control and silencing of virus replication in the brain and completely abolished the virus neurotropism. Furthermore, neuroinvasiveness of miRNA-targeted TBEV/LGTV viruses in very sensitive immunodeficient SCID mice was significantly limited. Immunocompetent animals immunized with such viruses were completely protected against challenge with the neurovirulent LGTV parent. These findings support the rationale of the miRNA-targeting approach to develop live attenuated virus vaccines against various neurotropic viruses.
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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, 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.
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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the role of mammalian reservoir hosts in Tick Borne Flavivirus biology
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Luwanika Mlera, Marshall E BloomAbstract:Small-to-medium sized mammals and large animals are lucrative sources of blood meals for ixodid Ticks that transmit life-threatening Tick-Borne Flaviviruses (TBFVs). TBFVs have been isolated from various organs obtained from wild-caught Myodes and Apodemus species in Europe and Asia. Thus, these rodents are well-established reservoirs of TBFVs. Wild-caught Peromyscus species have demonstrated seropositivity against Powassan virus, the only TBFV known to circulate in North America, suggesting that they may play an important role in the biology of the virus in this geographic region. However, virus isolation from Peromyscus species is yet to be demonstrated. Wild-caught medium-sized mammals, such as woodchucks (Marmota monax) and skunks (Mephitis mephitis) have also demonstrated seropositivity against POWV, and virus was isolated from apparently healthy animals. Despite the well-established knowledge that small-to-medium sized animals are TBFV reservoirs, specific molecular biology addressing host-pathogen interactions remain poorly understood. Elucidating these interactions will be critical for gaining insight into the mechanism(s) of viral pathogenesis and/or resistance.
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Peromyscus leucopus mouse brain transcriptome response to Powassan virus infection
Journal of NeuroVirology, 2018Co-Authors: Luwanika Mlera, Stephen F Porcella, Kimberly Meade-white, Eric Dahlstrom, Rachel Baur, Kishore Kanakabandi, Kimmo Virtaneva, Marshall E BloomAbstract:Powassan virus (POWV) is a Tick-Borne Flavivirus responsible for life-threatening encephalitis in North America and some regions of Russia. The Ticks that have been reported to transmit the virus belong to the Ixodes species, and they feed on small-to-medium-sized mammals, such as Peromyscus leucopus mice, skunks, and woodchucks. We previously developed a P. leucopus mouse model of POWV infection, and the model is characterized by a lack of clinical signs of disease following intraperitoneal or intracranial inoculation. However, intracranial inoculation results in mild subclinical encephalitis from 5 days post infection (dpi), but the encephalitis resolves by 28 dpi. We used RNA sequencing to profile the P. leucopus mouse brain transcriptome at different time points after intracranial challenge with POWV. At 24 h post infection, 42 genes were significantly differentially expressed and the number peaked to 232 at 7 dpi before declining to 31 at 28 dpi. Using Ingenuity Pathway Analysis, we determined that the genes that were significantly expressed from 1 to 15 dpi were mainly associated with interferon signaling. As a result, many interferon-stimulated genes (ISGs) were upregulated. Some of the ISGs include an array of TRIMs (genes encoding tripartite motif proteins). These results will be useful for the identification of POWV restriction factors.
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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, Dana P. Scott, Dan Long, Rebecca Rosenke, Tom G. Schwan, 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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Modeling Powassan virus infection in Peromyscus leucopus, a natural host
PLOS Neglected Tropical Diseases, 2017Co-Authors: Luwanika Mlera, Kimberly Meade-white, Greg Saturday, Dana P. Scott, Marshall E BloomAbstract:The Tick-Borne Flavivirus, Powassan virus (POWV) causes life-threatening encephalitis in humans in North America and Europe. POWV is transmitted by ixodid Tick vectors that feed on small to medium-sized mammals, such as Peromyscus leucopus mice, which may serve as either reservoir, bridge or amplification hosts. Intraperitoneal and intracranial inoculation of 4-week old Peromyscus leucopus mice with 103 PFU of POWV did not result in overt clinical signs of disease. However, following intracranial inoculation, infected mice seroconverted to POWV and histopathological examinations revealed that the mice uniformly developed mild lymphocytic perivascular cuffing and microgliosis in the brain and spinal cord from 5 to 15 days post infection (dpi), suggesting an early inflammatory response. In contrast, intracranial inoculation of 4-week old C57BL/6 and BALB/c mice was lethal by 5 dpi. Intraperitoneal inoculation was lethal in BALB/c mice, but 40% (2/5) of C57BL/6 mice survived. We concluded that Peromyscus leucopus mice infected i.c. with a lethal dose of POWV support a limited infection, restricted to the central nervous system and mount an antibody response to the virus. However, they fail to develop clinical signs of disease and are able to control the infection. These results suggest the involvement of restriction factors, and the mechanism by which Peromyscus leucopus mice restrict POWV infection remains under study.
Kentaro Yoshii - One of the best experts on this subject based on the ideXlab platform.
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trans complementation of replication defective omsk hemorrhagic fever virus for antiviral study
Virologica Sinica, 2019Co-Authors: Qiuyan Zhang, Kentaro Yoshii, Chenglin Deng, Zherui Zhang, Xiaodan Li, Hanqing Ye, Na Li, Bo ZhangAbstract:Omsk hemorrhagic fever virus (OHFV) is a Tick-Borne Flavivirus classified as a biosafety level-4 (BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion (OHFV-ΔNS1) and reporter virus replacing NS1 with the Gaussia luciferase (Gluc) (OHFV-ΔNS1-Gluc). Both the defective OHFV-ΔNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1 (BHK21NS1) but not in naive BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21NS1 cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z′ value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.
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detection of a novel Tick Borne Flavivirus and its serological surveillance
Ticks and Tick-borne Diseases, 2019Co-Authors: Hiroshi Shimoda, Kentaro Yoshii, Daisuke Hayasaka, Mayumi Yokoyama, Kazuo Suzuki, Yuuji Kodera, Tsutomu Takeda, Junko Mizuno, Keita Noguchi, Kenzo YonemitsuAbstract:Abstract Tick-Borne encephalitis virus (TBEV), a Flavivirus that causes severe neurological symptoms in humans, has been found in Hokkaido, Japan. In the present study, we detected sequences from a novel Tick-Borne Flavivirus, designated Yamaguchi virus (YGV), in liver and serum samples obtained from a wild boar in the Yamaguchi prefecture, Japan. Phylogenetic analysis revealed that YGV belongs to the TBEV complex and is closely related to Langat virus (LGTV). YGV was also detected by specific RT-PCR from 20 in 378 pools of Ticks (2923 Ticks) collected in Yamaguchi and Wakayama prefectures and from seven in 46 wild boar captured in Wakayama. The major Ticks infected with YGV belong to the genus Haemaphysalis. Unfortunately, YGV could not be isolated from any samples from the RT-PCR positive wild boar or Ticks. Therefore, ELISA for detection of antibodies against YGV was established using LGTV, and surveillance was performed among wild boar in 10 different prefectures on Honshu Island, the main island of Japan. The results showed that the seroprevalence of Tick-Borne Flavivirus infection in the Wakayama and Hyogo prefectures of western Japan was significantly higher than that in the other prefectures, while antibodies against Tick-Borne Flavivirus were not detected in any wild boar in the Tochigi prefecture in the eastern part of Japan. In addition, wild raccoons or masked palm civets in the Hyogo prefecture did not possess detectable antibodies against Tick-Borne Flaviviruses. In conclusion, YGV appears to be maintained primarily among wild boar and Ticks in the western part of Japan. YGV is the second Flavivirus (after Japanese encephalitis virus) shown to be circulating on Honshu Island in Japan.
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Tick-Borne encephalitis
Uirusu, 2017Co-Authors: Kentaro YoshiiAbstract:Tick-Borne encephalitis virus (TBEV) belongs to the Flaviviridae family and Flavivirus genus. TBEV is maintained in transmission cycles between Ixodid Ticks and wild mammalian hosts, particularly rodents. A wide range of animal species are also infected with TBEV by the bite of infected Ticks, and TBEV infection causes fatal encephalitis in humans. TBEV is endemic widely in the Eurasian continent, and more than 10,000 cases of the disease are reported annually. In Japan, the 1st confirmed case of TBE was reported in the southern area of Hokkaido in 1993, and after 20 years, the 2nd to 4th cases were reported in Hokkaido in 2016 and 2017. Our sero-epizootiological survey indicated endemic foci of TBEV are widely distributed in Hokkaido and that those of TBEV or Tick-Borne Flavivirus outside Hokkaido. In this review, I introduced recent topics of TBEV including newly developed diagnostic methods, epidemiology and pathogenesis of TBEV.
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dendritic transport of Tick Borne Flavivirus rna by neuronal granules affects development of neurological disease
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Minato Hirano, Hiroaki Kariwa, Memi Muto, Mizuki Sakai, Hirofumi Kondo, Shintaro Kobayashi, Kentaro YoshiiAbstract:Abstract Neurological diseases caused by encephalitic Flaviviruses are severe and associated with high levels of mortality. However, little is known about the detailed mechanisms of viral replication and pathogenicity in the brain. Previously, we reported that the genomic RNA of Tick-Borne encephalitis virus (TBEV), a member of the genus Flavivirus, is transported and replicated in the dendrites of neurons. In the present study, we analyzed the transport mechanism of the viral genome to dendrites. We identified specific sequences of the 5′ untranslated region of TBEV genomic RNA that act as a cis-acting element for RNA transport. Mutated TBEV with impaired RNA transport in dendrites caused a reduction in neurological symptoms in infected mice. We show that neuronal granules, which regulate the transport and local translation of dendritic mRNAs, are involved in TBEV genomic RNA transport. TBEV genomic RNA bound an RNA-binding protein of neuronal granules and disturbed the transport of dendritic mRNAs. These results demonstrated a neuropathogenic virus hijacking the neuronal granule system for the transport of viral genomic RNA in dendrites, resulting in severe neurological disease.
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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, Emmanuel Pacia Hernandez, Kozo Fujisaki, Remil Linggatong Galay, Masami Mochizuki, 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.
Michael J. Turell - One of the best experts on this subject based on the ideXlab platform.
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Experimental Transmission of Karshi (Mammalian Tick-Borne Flavivirus Group) Virus by Ornithodoros Ticks >2,900 Days after Initial Virus Exposure Supports the Role of Soft Ticks as a Long-Term Maintenance Mechanism for Certain Flaviviruses.
PLOS Neglected Tropical Diseases, 2015Co-Authors: Michael J. TurellAbstract:Background Members of the mammalian Tick-Borne Flavivirus group, including Tick-Borne encephalitis virus, are responsible for at least 10,000 clinical cases of Tick-Borne encephalitis each year. To attempt to explain the long-term maintenance of members of this group, we followed Ornithodoros parkeri, O. sonrai, and O. tartakovskyi for >2,900 days after they had been exposed to Karshi virus, a member of the mammalian Tick-Borne Flavivirus group. Methodology/Principal Findings Ticks were exposed to Karshi virus either by allowing them to feed on viremic suckling mice or by intracoelomic inoculation. The Ticks were then allowed to feed individually on suckling mice after various periods of extrinsic incubation to determine their ability to transmit virus by bite and to determine how long the Ticks would remain infectious. The Ticks remained efficient vectors of Karshi virus, even when tested >2,900 d after their initial exposure to virus, including those Ticks exposed to Karshi virus either orally or by inoculation. Conclusions/Significance Ornithodoros spp. Ticks were able to transmit Karshi virus for >2,900 days (nearly 8 years) after a single exposure to a viremic mouse. Therefore, these Ticks may serve as a long-term maintenance mechanism for Karshi virus and potentially other members of the mammalian Tick-Borne Flavivirus group.
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experimental transmission of karshi mammalian Tick Borne Flavivirus group virus by ornithodoros Ticks 2 900 days after initial virus exposure supports the role of soft Ticks as a long term maintenance mechanism for certain Flaviviruses
PLOS Neglected Tropical Diseases, 2015Co-Authors: Michael J. TurellAbstract:Background Members of the mammalian Tick-Borne Flavivirus group, including Tick-Borne encephalitis virus, are responsible for at least 10,000 clinical cases of Tick-Borne encephalitis each year. To attempt to explain the long-term maintenance of members of this group, we followed Ornithodoros parkeri, O. sonrai, and O. tartakovskyi for >2,900 days after they had been exposed to Karshi virus, a member of the mammalian Tick-Borne Flavivirus group. Methodology/Principal Findings Ticks were exposed to Karshi virus either by allowing them to feed on viremic suckling mice or by intracoelomic inoculation. The Ticks were then allowed to feed individually on suckling mice after various periods of extrinsic incubation to determine their ability to transmit virus by bite and to determine how long the Ticks would remain infectious. The Ticks remained efficient vectors of Karshi virus, even when tested >2,900 d after their initial exposure to virus, including those Ticks exposed to Karshi virus either orally or by inoculation. Conclusions/Significance Ornithodoros spp. Ticks were able to transmit Karshi virus for >2,900 days (nearly 8 years) after a single exposure to a viremic mouse. Therefore, these Ticks may serve as a long-term maintenance mechanism for Karshi virus and potentially other members of the mammalian Tick-Borne Flavivirus group.
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assay for and replication of karshi mammalian Tick Borne Flavivirus group virus in mice
American Journal of Tropical Medicine and Hygiene, 2008Co-Authors: Michael J. Turell, Chris A Whitehouse, Ashley Butler, Carson Baldwin, Hannah Hottel, Christopher N MoresAbstract:Little is known about the replication of Karshi virus, a member of the mammalian Tick-Borne Flavivirus group, in its rodent hosts. Therefore, we developed a quantitative real-time RT-PCR assay and measured the amount of viral RNA in selected tissues of infected Swiss Webster mice. Two-day-old mice were highly susceptible, with 100% fatality 9 to 12 days after infection, whereas infection in 9-day-old mice was less virulent, with death occurring only rarely. In nearly all cases, mice inoculated when 2 days old contained similar numbers of viral genome equivalents from blood and liver samples from any given mouse, with titers declining after day 7. In contrast, the amount of viral RNA in the brain began to rise rapidly 4 days after exposure, peaked at about 6 days after virus exposure (titer of > 10 13 genome equivalents/g), and remained at that level until euthanasia or death. Viral profiles were similar in needle- inoculated or Tick-exposed mice.
Michael R Holbrook - One of the best experts on this subject based on the ideXlab platform.
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Kyasanur Forest disease virus infection activates human vascular endothelial cells and monocyte-derived dendritic cells
Emerging microbes & infections, 2018Co-Authors: Jana Širmarová, Michael R Holbrook, Jiri Salat, Martin Palus, Václav Hönig, Helena Langhansová, Daniel RuzekAbstract:Kyasanur Forest disease virus (KFDV) is a highly pathogenic Tick-Borne Flavivirus enzootic to India. In humans, KFDV causes a severe febrile disease. In some infected individuals, hemorrhagic manifestations, such as bleeding from the nose and gums and gastrointestinal bleeding with hematemesis and/or blood in the stool, have been reported. However, the mechanisms underlying these hemorrhagic complications remain unknown, and there is no information about the specific target cells for KFDV. We investigated the interaction of KFDV with vascular endothelial cells (ECs) and monocyte-derived dendritic cells (moDCs), which are key targets for several other hemorrhagic viruses. Here, we report that ECs are permissive to KFDV infection, which leads to their activation, as demonstrated by the upregulation of E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 at the mRNA and protein levels. Increased expression of these adhesive molecules correlated with increased leukocyte adhesion. Infected ECs upregulated the expression of interleukin (IL)-6 but not IL-8. Additionally, moDCs were permissive to KFDV infection, leading to increased release of IL-6 and tumor necrosis factor-α. Supernatants from KFDV-infected moDCs caused EC activation, as measured by leukocyte adhesion. The results indicate that ECs and moDCs can be targets for KFDV and that both direct and indirect mechanisms can contribute to EC activation.
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Comparative pathogenesis of Alkhumra hemorrhagic fever and Kyasanur forest disease viruses in a mouse model.
PLOS Neglected Tropical Diseases, 2014Co-Authors: Bevan Sawatsky, Michael R Holbrook, Alexander J. Mcauley, Dennis A. BenteAbstract:Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV) are genetically closely-related, Tick-Borne Flaviviruses that cause severe, often fatal disease in humans. Flaviviruses in the Tick-Borne encephalitis (TBE) complex typically cause neurological disease in humans whereas patients infected with KFDV and AHFV predominately present with hemorrhagic fever. A small animal model for KFDV and AHFV to study the pathogenesis and evaluate countermeasures has been lacking mostly due to the need of a high biocontainment laboratory to work with the viruses. To evaluate the utility of an existing mouse model for Tick-Borne Flavivirus pathogenesis, we performed serial sacrifice studies in BALB/c mice infected with either KFDV strain P9605 or AHFV strain Zaki-1. Strikingly, infection with KFDV was completely lethal in mice, while AHFV caused no clinical signs of disease and no animals succumbed to infection. KFDV and high levels of pro-inflammatory cytokines were detected in the brain at later time points, but no virus was found in visceral organs; conversely, AHFV Zaki-1 and elevated levels of cytokines were found in the visceral organs at earlier time points, but were not detected in the brain. While infection with either virus caused a generalized leukopenia, only AHFV Zaki-1 induced hematologic abnormalities in infected animals. Our data suggest that KFDV P9605 may have lost its ability to cause hemorrhagic disease as the result of multiple passages in suckling mouse brains. However, likely by virtue of fewer mouse passages, AHFV Zaki-1 has retained the ability to replicate in visceral organs, cause hematologic abnormalities, and induce pro-inflammatory cytokines without causing overt disease. Given these striking differences, the use of inbred mice and the virus passage history need to be carefully considered in the interpretation of animal studies using these viruses.
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A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice
Journal of Virology, 2014Co-Authors: Kentaro Yoshii, Michael R Holbrook, Hiroaki Kariwa, Manabu Igarashi, Yuji Sunden, Kana Yokozawa, Ikuo TakashimaAbstract:Tick-Borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic Tick-Borne Flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of Tick-Borne Flaviviruses. IMPORTANCE Tick-Borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) belong to the Tick-Borne encephalitis serocomplex, genus Flavivirus, family Flaviviridae. Although TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. In this study, we investigated the viral determinants responsible for the different disease phenotypes using reverse genetics technology. We identified a cluster of only four amino acids in nonstructural protein 5 primarily involved in the development of neurological disease in a mouse model. Moreover, the effect of these four amino acids was independent of viral replication property and did not affect the formation of virus-induced lesions in the brain directly. These data suggest that these amino acids may be involved in the induction of neuronal dysfunction and degeneration in virus-infected neurons, ultimately leading to the neurological disease phenotype. These findings provide new insight into the molecular mechanisms of Tick-Borne Flavivirus pathogenesis.
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Clinical evaluation of highly pathogenic Tick-Borne Flavivirus infection in the mouse model.
Journal of Medical Virology, 2009Co-Authors: Bersabeh Tigabu, Terry L Juelich, Joseph Bertrand, Michael R HolbrookAbstract:The objective of this study was to evaluate the feasibility of using clinical parameters to demonstrate disease progression and differentiate between Omsk hemorrhagic fever virus (OHFV) and Russian spring-summer encephalitis virus (RSSEV) infection in the mouse model. Adult C57BL/6 and balb/c mice were infected with either OHFV or RSSEV by footpad inoculation and their temperature, body weight, clinical signs complete blood count, and blood chemistries were evaluated for up to 15 days post-infection (dpi). Clinical evaluation showed that OHFV infection seriously affects balb/c mice, which had shorter average survival times (ASTs) than other groups. On the contrary, RSSEV infection of C57BL/6 mice was more severe than in balb/c mice. During these studies, the development of fever was not observed and the body weight of OHFV infected balb/c and C57BL/6 mice began to decline sharply starting from day 7 and 8, respectively, which correlated with disease onset. Peak increase of globulin and neutrophils was demonstrated after 9 dpi in OHFV infected mice; however, the lymphocyte number was not affected. Viremia was undetectable in these animals with either virus infection, but virus was found in most organs tested. These results indicate marked differences in the clinical signs, pathology, and immune response of mice infected with either OHFV or RSSEV and further validate the use of this mouse model system to evaluate human disease. J. Med. Virol. 81:1261–1269, 2009. © 2009 Wiley-Liss, Inc.
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Use of Recombinant E Protein Domain III-Based Enzyme-Linked Immunosorbent Assays for Differentiation of Tick-Borne Encephalitis Serocomplex Flaviviruses from Mosquito-Borne Flaviviruses
Journal of Clinical Microbiology, 2004Co-Authors: Michael R Holbrook, Robert E Shope, Alan D.t. BarrettAbstract:The serological diagnosis of infection by Flaviviruses is complicated by the presence of Flavivirus cross-reactive antibodies that produce false-positive results for Flavivirus infections, especially in regions where more than one virus is endemic. Current diagnostic reagents for Tick-Borne Flavivirus infection have been found to cross-react with yellow fever- or dengue virus-positive sera. This study utilized recombinant Flavivirus E protein domain 3 (rE-D3) as a diagnostic reagent to differentiate between infection by mosquito- and Tick-Borne Flaviviruses. This study found that the use of rE-D3 in an enzyme-linked immunosorbent assay (ELISA)-based format allowed the differentiation between serum specific for either mosquito- or Tick-Borne Flaviviruses, but not among the members of the Tick-Borne encephalitis (TBE) serocomplex of Flaviviruses. Sera derived against several TBE serocomplex rE-D3 were found to cross-react with heterologous rE-D3 within the TBE serocomplex, but not with those from mosquito-Borne Flaviviruses, in both Western blots and ELISAs. Mouse hyperimmune sera generated against TBE serocomplex viruses were also found to react specifically with TBE serocomplex rE-D3, but not with rE-D3 from mosquito-Borne viruses and vice versa. When a similar test using virus-derived antigen was performed, a loss of both specificity and sensitivity was observed. These results indicate that Flavivirus rE-D3 would be a useful reagent for the detection of infection by TBE serocomplex Flaviviruses, several of which are potential biothreat agents, but would not provide the ability to differentiate among infections by separate members of the serocomplex.