Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay

Zhang, Pan Tao; Shan, Chao; Li, Xiao Dan; Liu, Si Qing; Deng, Cheng; Ye, Han-Qing; Shang, Bao Di; Shi, Pei Yong; Lv, Ming; Shen, Bei Fen; Cheng, Qi; Zhang, Bo

doi:10.1016/j.virusres.2015.09.015

Cited by 25 publications

(26 citation statements)

References 30 publications

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“…However, the instability or decreased fitness of the recombinant viruses compared to the parental virus limit their application in some experimental designs, as noted by Aubry et al (2015). This instability in engineered virus expressing reporter genes was also reported in other flavivirus and DNA virus platforms (Song et al 2012, Vandergaast et al 2014, Zhang et al 2015.…”

Section: Discussionsupporting

confidence: 61%

“…YFV, GFP, and firefly and Renilla luciferases) have been reported for distinct viruses and used for various purposes, e.g. on the evaluation of virus-host interactions, viral tropism, and antiviral and neutralization assays (Roth et al 2009, Schoggins et al 2012, van den Worm et al 2012, Dag et al 2013, De Baets et al 2015, Zhang et al 2015. These studies include the development of recombinant flavivirus expressing reporter genes, such as Dengue virus expressing GFP and firefly and Renilla luciferases (Zou et al 2011, Schoggins et al 2012.…”

Section: Discussionmentioning

confidence: 99%

“…A desired feature of such clones is the stability of GLuc expression to allow their use in long-term studies, but unfortunately such condition has not always been achieved. Zhang et al (2015) reported the construction of a West Nile Virus (WNV) expressing GLuc and its use in neutralization assays, but the recombinant virus gradually lost stability along the passages, where GLuc activity was no longer detected in passage 3. Vandergaast et al (2014) also found similar instability in a GLuc-expressing WNV.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, infectious clones or replicon systems expressing reporter genes (e.g. firefly and Renilla luciferases, green and yellow fluorescent proteins) have been developed and used mainly on molecular virology studies, to construct viral vectors and on sophisticated antiviral assays (De Baets et al 2015, Patkar et al 2009, Roth et al 2009, Schoggins et al 2012, van den Worm et al 2012, Dag et al 2013, Kato et al 2014, Ding et al 2015, Zhang et al 2015. Among the reporter genes, Gaussia luciferase (GLuc) started to be used in in vivo and in vitro assays due to some advantages such as high stability and sensitivity, and the fact that it is secreted, facilitating its detection in several experiments (Tannous et al 2005, Venisnik et al 2007.…”

Section: Yfv Is Endemic In Countries Of Africa and Southmentioning

confidence: 99%

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Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

Kassar

Magalhães

Júnior

et al. 2017

An. Acad. Bras. Ciênc.

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Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r 2 =0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.

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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Yfv Is Endemic In Countries Of Africa and Southmentioning

confidence: 99%

See 2 more Smart Citations

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

Kassar

Magalhães

Júnior

et al. 2017

An. Acad. Bras. Ciênc.

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“…A possible explanation of the lack of association between intensity of radiance and recovery of infectious virus is the expression characteristics of the luciferase gene in the MCMV genome. The luciferase coding sequence was placed under the control of the M78 promoter, a gene with early kinetics (Oliveira & Shenk, 2001 it is probable that radiance signal would be better associated proportionally associated with viral production (Zhang et al, 2016a). Nonetheless, as our main objective was to study viral dissemination, regulation of luciferase expression by a promoter of an early gene serves as a more sensitive method to detect sites of viral spread.…”

Section: M33 Role In MCMV Disseminationmentioning

confidence: 99%

Cell-type specific activities of cytomegalovirus GPCR homologues

Oliveira¹

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The human cytomegalovirus (HCMV) is the main agent of congenital infection worldwide. Although primary infections or reactivations do not represent a threat to most individuals, they impose a lifethreating event to a developing foetus or immunocompromised individuals. Although we have learnt much about sites of viral replication and cells involved in the end-stage disease, the specific pathway and essential cell types that the virus must go through to later establish a successful infection and latency is still not clear. HCMV encodes four GPCR homologues (vGPCR): US27, US28, UL33 and UL78. vGPCR have been implicated in viral pathogenesis due to their ability to activate signalling pathways and, thus, alter physiological processes to favour infection. US28 and UL33 have been shown to activate several kinases and transcriptional factors. A key component of both US28 and UL33 sequence is the DRY (Asp/Arg/Tyr) motif, a region directly involved with G protein binding.Because Gα protein content is cell type-dependent, signalling activation by vGPCR can differ from cell to cell. Furthermore, these receptors share the particular characteristic of being constitutively Functional studies demonstrated migration induction of HTR-8 following transfection by either US28 or UL33. In order to investigate potential mechanisms, signalling pathways were probed via detection of activated kinases and the secretion of matrix metalloproteinases (MMP) and chemokines (CK) via luminex assays. Although differences in the level of kinases could not be measured by western blot, induction of MMP and CK were detected by Luminex assays, with contrasting results for US28 and UL33 in transfected cells (HTR-8 and HUVEC). An HCMV recombinant disrupted for US28 signalling (DQY) was generated and compared with wild type HCMV and a US28 null mutant for induction of MMP and CK in virus-infected cells. US28-dependent effects were identified for infected human foreskin fibroblasts, but results for HUVEC were equivocal.iii To determine potential bottlenecks to viral dissemination that may require M33 function(s), mice were infected with M33 knockout (M33) viruses by different routes: intranasal, -mimicking the most natural route, footpad -a more compartmentalized route, and intraperitoneal, the most direct route to major organs. To help track viral dissemination, a luciferase tagged M33 was generated and infection was compared to control virus (M78luc). M33 intranasal infection did not result in attenuation for colonisation of the nose, draining (superficial cervical) lymph nodes (dLN) or the lungs. Via footpad route, M33 colonised the footpad and dLN to levels equivalent to control virus, but was significantly attenuated in spread to the spleen and, subsequently, the salivary glands.Following intraperitoneal infection, the virus is readily delivered to major organs due to direct access to the bloodstream, there was no difference in the colonisation of primary organs (spleen, liver) between M33 and control MCMV, but M33 was still unable ...

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