The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles

Lu, Chien-Yi; Lin, Chensheng; Lai, Hsueh‐Chou; Yang, Yu; Liao, Chih‐Yi; Su, Wu‐Chung; Ko, Bo-Han; Chang, Young-Sheng; Huang, Su-Hua; Lin, Cheng‐Wen

doi:10.3390/v11111005

Cited by 9 publications

(19 citation statements)

References 31 publications

(45 reference statements)

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“…The length of the transcript RNA was confirmed using RNA electrophoresis ( Figure 2B ). We used a DL5000 DNA marker, and the length of the target RNA was expected to be 2,000–3,000bp, according to a previous study ( Lu et al, 2019 ).…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, there should be an HDVr sequence followed by a polymerase II terminator and a polyadenylation signal (pA) at the 3' end. The plasmids are directly transfected into susceptible cells to produce the recovery virus ( Schwarz et al, 2016 ; Lu et al, 2019 ) (iii) The third method uses infectious subgenomic amplicons (ISA) that separate the virus genome into 3–4 fragments with overlapping sequences and clone them into plasmids, respectively. After co-transfection and in vivo homologous recombination, full-length cDNA clones are generated inside the susceptible cells ( Atieh et al, 2017 ; Touret et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

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Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Qin

Chen

et al. 2021

Front. Microbiol.

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Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Qin

Chen

et al. 2021

Front. Microbiol.

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“…The assay was further followed by measuring ZIKV positive-strand RNA genomes using SYBR Green Master Mix kit with NS5-specific primer pairs (5’-CTTGTGGCTGCTGCGGAGGTCA-3’ and 5’-GTGGTGGGAGCAAAACGGAACTT-3’) by 7300 Realtime PCR system (Applied Biosystems, Foster City, CA, USA). The corresponding threshold cycle value ( C t ) measured was determined the relative levels of positive- and negative- strand RNA genomes in (un-)treated infected cells were determined by real-time RT-PCR assay normalized by the housekeeping gene GAPDH, described in our prior report [ 25 ]. In addition, the infected/treated cells were harvested 72 h post incubation for assessing intracellular infectious viral particles, and then lysed through three freeze-thaw cycles.…”

Section: Methodsmentioning

confidence: 99%

Effective Antiviral Activity of the Tyrosine Kinase Inhibitor Sunitinib Malate against Zika Virus

et al. 2021

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Introduction Zika virus (ZIKV), a mosquito-borne flavivirus, causes the outbreaks of Latin America in 2015 - 2016, with the incidence of neurological complications. Sunitinib malate, an orally bioavailable malate salt of the tyrosine kinase inhibitor, is suggested as a broad-spectrum antiviral agent against emerging viruses like severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Materials and Methods This study investigated the antiviral efficacy and antiviral mechanisms of sunitinib malate against ZIKV infection using cytopathic effect reduction, virus yield, and time-of-addition assays. Results Sunitinib malate concentration-dependently reduced ZIKV-induced cytopathic effect, the expression of viral proteins, and ZIKV yield in supernatant with 50% inhibitory concentration (IC 50 ) value of 0.015 μM, and the selectivity index of greater than 100 against ZIKV infection, respectively. Sunitinib malate had multiple antiviral actions during entry and post-entry stages of ZIKV replication. Sunitinib malate treatment at entry stage significantly reduced the levels of ZIKV RNA replication with the reduction of (+) RNA to (-) RNA ratio and the production of new intracellular infectious particles in infected cells. The treatment at post-entry stage caused a concentration-dependent increase in the levels of ZIKV (+) RNA and (-) RNA in infected cells, along with enlarging the ratio of (+) RNA to (-) RNA, but caused a pointed increase in the titer of intracellular infectious particles by 0.01 and 0.1 μM, and a substantial decrease in the titer of intracellular infectious particles by 1 μM. Conclusion The study discovered the antiviral actions of sunitinib malate against ZIKV infection, demonstrating a repurposed, host-targeted approach to identify potential antiviral drugs for treating emerging and global viral diseases.

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“…The creation of recombinant viruses with specific mutations through the manipulation of the ZIKV genome has enabled researchers to shed a light on the function of viral proteins, the interactions involved between ZIKV and its host, and associated disease [ 36 ]. Research strategies harnessing reverse genetics have also proven valuable in the development of new effective prevention and control measures [ 37 , 38 ]. The bioengineering of replicating-competent recombinant ZIKV, which can be used to modify the ZIKV genomic RNA at the DNA level and express reporter genes, has, in fact, shown remarkable potential for the development of anti-viral therapeutic options to treat ZIKV infection [ 39 , 40 ], provided that the instability commonly linked to the construction of full-length cDNA clones can be overcome [ 41 , 42 ].…”

Section: Reverse Genetics and Recombinant Zikvmentioning

confidence: 99%

Congenital Zika Syndrome: Genetic Avenues for Diagnosis and Therapy, Possible Management and Long-Term Outcomes

Gullo

Scaglione

Cucinella

et al. 2022

JCM

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Zika virus (ZIKV) was discovered in Uganda in 1947 and was originally isolated only in Africa and Asia. After a spike of microcephaly cases in Brazil, research has closely focused on different aspects of congenital ZIKV infection. In this review, we evaluated many aspects of the disease in order to build its natural history, with a focus on the long-term clinical and neuro-radiological outcomes in children. The authors have conducted a wide-ranging search spanning the 2012–2021 period from databases PubMed, PubMed Central, Web of Science, Medline, Scopus. Different sections reflect different points of congenital ZIKV infection syndrome: pathogenesis, prenatal diagnosis, clinical signs, neuroimaging and long-term developmental outcomes. It emerged that pathogenesis has not been fully clarified and that the clinical signs are not only limited to microcephaly. Given the current absence of treatments, we proposed schemes to optimize diagnostic protocols in endemic countries. It is essential to know the key aspects of this disease to guarantee early diagnosis, even in less severe cases, and an adequate management of the main chronic problems. Considering the relatively recent discovery of this congenital infectious syndrome, further studies and updated long-term follow-up are needed to further improve management strategies for this disease.

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The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles

Cited by 9 publications

References 31 publications

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Effective Antiviral Activity of the Tyrosine Kinase Inhibitor Sunitinib Malate against Zika Virus

Congenital Zika Syndrome: Genetic Avenues for Diagnosis and Therapy, Possible Management and Long-Term Outcomes

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