Chimeric Tick-Borne Encephalitis/Dengue Virus Is Attenuated in Ixodes scapularis Ticks and Aedes aegypti Mosquitoes

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Vector borne and zoonotic diseases (Larchmont, N.Y.) (Impact Factor: 2.3). 12/2010; 11(6):665-74. DOI: 10.1089/vbz.2010.0179
Source: PubMed


In an effort to derive an efficacious live attenuated vaccine against tick-borne encephalitis, we generated a chimeric virus bearing the structural protein genes of a Far Eastern subtype of tick-borne encephalitis virus (TBEV) on the genetic background of recombinant dengue 4 (DEN4) virus. Introduction of attenuating mutations into the TBEV envelope protein gene, as well as the DEN4 NS5 protein gene and 3' noncoding region in the chimeric genome, results in decreased neurovirulence and neuroinvasiveness in mice, and restricted replication in mouse brain. Since TBEV and DEN4 viruses are transmitted in nature by ticks and mosquitoes, respectively, it was of interest to investigate the infectivity of the chimeric virus for both arthropod vectors. Therefore, parental and chimeric viruses were tested for growth in mosquito and tick cells and for oral infection in vivo. Although all chimeric viruses demonstrated moderate levels of replication in C6/36 mosquito cells, they were unable to replicate in ISE6 tick cells. Further, the chimeric viruses were unable to infect or replicate in Aedes aegypti mosquitoes and Ixodes scapularis tick larvae. The poor infectivity for both potential vectors reinforces the safety of chimeric virus-based vaccine candidates for the environment and for use in humans.

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Available from: Marshall Bloom, Mar 10, 2014
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    • "The construction and characterization of chimeric flaviviruses has also provided critical information on the genetic elements that modulate the differential vector ranges, transmissibilities and disease phenotypes of divergent flaviviruses. Several of these studies have been performed using representative flaviviruses from the tick-borne and mosquito-borne groups [39–44]. More pertinent to this investigation are the few studies that describe the construction and characterization of viral chimeras between NKV and arthropod/vertebrate flaviviruses [45–47]. "
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    ABSTRACT: Background Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. Methods Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. Results Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. Conclusions Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.
    Virology Journal 08/2014; 11(1):150. DOI:10.1186/1743-422X-11-150 · 2.18 Impact Factor
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    • "A number of flavivirus chimeric virus studies have indicated that regions outside the prME envelope genes modulate infection of mosquito cells in vitro (Charlier et al., 2010; Johnson et al., 2003, 2004; Pletnev & Men, 1998) and in in vitro mosquito models (Brault et al., 2011; Hanley et al., 2005). However, some chimeric flavivirus studies also implicate structural regions as contributing determinants for infection of the mosquito vector (Engel et al., 2011; Pletnev, Bray, Huggins, & Lai, 1992). In vitro experiments in C6/36 cells with prME chimeras between WNV and St. Louis encephalitis virus (SLEV) suggest that portions of both the nonstructural and structural gene regions are determinants for vector infection; however, the same chimeric viruses compared in Cx. tarsalis cells indicated that structural genes were the primary determinants (Maharaj et al., 2012). "
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    ABSTRACT: Arthropod-borne viruses (arboviruses) are transmitted between vertebrate hosts and arthropod vectors. An inherently complex interaction among virus, vector, and the environment determines successful transmission of the virus. Once believed to be "flying syringes," recent advances in the field have demonstrated that mosquito genetics, microbiota, salivary components, and mosquito innate immune responses all play important roles in modulating arbovirus transmissibility. The literature on the interaction among virus, mosquito, and environment has expanded dramatically in the preceding decade and the utilization of next-generation sequencing and transgenic vector methodologies assuredly will increase the pace of knowledge acquisition in this field. This chapter outlines the interplay among the three factors in both direct physical and biochemical manners as well as indirectly through superinfection barriers and altered induction of innate immune responses in mosquito vectors. The culmination of the aforementioned interactions and the arms race between the mosquito innate immune response and the capacity of arboviruses to antagonize such a response ultimately results in the subjugation of mosquito cells for viral replication and subsequent transmission.
    Advances in Virus Research 04/2014; 89:39-83. DOI:10.1016/B978-0-12-800172-1.00002-1 · 4.57 Impact Factor
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    • "Nevertheless, the necessity for revaccinations every 3–5 years, probability of allergic reactions in recipients, and rare registration of TBE cases among vaccine recipients [8]–[9] have necessitated the development of new approaches to design new prophylactic preparations. During recent years several studies have been conducted to develop new generation vaccines against TBE [10]–[20]. There are no drugs for emergency prophylaxis and treatment of TBE, although the research studies based on various approaches are being conducted [21]–[24]. "
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    ABSTRACT: Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8-16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.
    PLoS ONE 04/2013; 8(4):e61094. DOI:10.1371/journal.pone.0061094 · 3.23 Impact Factor
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