Improved genetic stability of recombinant yellow fever 17D virus expressing a lentiviral Gag gene fragment

Santana, Marlon G. Veloso de; Neves, Patrícia; Santos, Juliana Ribeiro dos; Lima, Noemia S.; Santos, Alexandre Araújo Cunha dos; Watkins, David I.; Galler, Ricardo; Bonaldo, Myrna C.

doi:10.1016/j.virol.2014.01.017

Cited by 13 publications

(8 citation statements)

References 42 publications

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“…We infected midgut, salivary gland, and synganglion cultures with LGTV expressing green fluorescent protein (LGTV GFP ) and observed the cultures by live fluorescence microscopy, using the GFP signal as an indicator of virus infection and gene expression. The growth of recombinant flaviviruses may differ from parental viruses (50,51); however, the potential difference in growth between LGTV GFP and its wild-type LGTV TP21 strain in these organs has not been determined.…”

Section: Resultsmentioning

confidence: 99%

Flavivirus Infection ofIxodes scapularis(Black-Legged Tick)Ex VivoOrganotypic Cultures and Applications for Disease Control

Grabowski¹,

Tsetsarkin

Long³

et al. 2017

mBio

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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. LGTVand 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,

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Section: Resultsmentioning

confidence: 99%

Flavivirus Infection ofIxodes scapularis(Black-Legged Tick)Ex VivoOrganotypic Cultures and Applications for Disease Control

Grabowski¹,

Tsetsarkin

Long³

et al. 2017

mBio

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“…69 We managed to substantially improve the genetic stability of this recombinant YF17D virus by introducing mutations in the IRES element. 70 The foreign antigens expressed by the recombinant YF17D virus in the E/NS1 region appear to remain cell-associated as described elsewhere. 56,65,66 The heterologous proteins are indeed retained in the ER compartment due to the presence of anchor transmembrane motifs at their carboxi-terminus.…”

Section: Expression Of Larger Fragments By the Yf17d Virusmentioning

confidence: 99%

The yellow fever 17D virus as a platform for new live attenuated vaccines

Bonaldo

Sequeira

Galler³

2014

Human Vaccines & Immunotherapeutics

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“…Improved genetic stability of rYFV17D is also important to express large inserts in rYFV17D. Others have demonstrated that an insertion region RNA was detected up to twenty serial passages in Vero cells (de Santana et al, 2014). In spite of increase in viral genetic stability, these changes did not lead to an increase in immunogenicity in a mouse model by modifying IRES elements at the 5′ end of the SIV gag gene (de Santana et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…Others have demonstrated that an insertion region RNA was detected up to twenty serial passages in Vero cells (de Santana et al, 2014). In spite of increase in viral genetic stability, these changes did not lead to an increase in immunogenicity in a mouse model by modifying IRES elements at the 5′ end of the SIV gag gene (de Santana et al, 2014). The inserts of EGFP (green fluorescent protein variant 238 amino acids) (Bonaldo et al, 2007) and HIV p24 capsid protein (231 amino acids) (Franco et al, 2010) have been studied by others, while here, the CH505TF gp120 has an insert size encoding 458 amino acids (Liao et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Development of a recombinant yellow fever vector expressing a HIV clade C founder envelope gp120

Liao

Pritchett

et al. 2017

Journal of Virological Methods

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Development of a HIV-1 vaccine is a major global priority. The yellow fever virus (YFV) attenuated vaccine 17D is among the most effective of currently used vaccines. However, the stability of the YFV17D vector when carrying non-flavivirus genes has been problematic. We have constructed and expressed HIV-1 Env in YFV17D with either single transmembrane (STM) or double transmembrane (DTM) YFV E protein domains for the development of anti-HIV antibodies. Here we describe modifications of the YFV17D vector such that HIV-1 Env gp120 is expressed in up to 5 passages in Vero cells. Immunization with recombinant YFV17D vector prime followed by HIV-1 CH505 TF gp120 protein boosts were able to induce neutralizing antibodies for a HIV-1 tier 1 isolate in mice. This modified YFV vector may be a starting point for constructing HIV-1 vaccine candidate priming vectors.

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Improved genetic stability of recombinant yellow fever 17D virus expressing a lentiviral Gag gene fragment

Cited by 13 publications

References 42 publications

Flavivirus Infection ofIxodes scapularis(Black-Legged Tick)Ex VivoOrganotypic Cultures and Applications for Disease Control

Flavivirus Infection ofIxodes scapularis(Black-Legged Tick)Ex VivoOrganotypic Cultures and Applications for Disease Control

The yellow fever 17D virus as a platform for new live attenuated vaccines

Development of a recombinant yellow fever vector expressing a HIV clade C founder envelope gp120

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