Figure 1 - available from: Scientific Reports
This content is subject to copyright. Terms and conditions apply.
Schematic diagram of full-length ZIKV infectious cDNA clone (a) Artifical synthetic DNA sequences 1&2 that contain ZIKV cDNA and in vitro transcription elements (T7 promoter and Ribozyme). Full-length ZIKA cDNA was designed to construct into pFK vector by using indicated restriction enzymes (Sbf1/Afe1/ Mlu1). (b) Artificial synthetic cDNA sequences 1&2 were inserted into low-copy plasmid pFK, respectively. After transformation, ten colonies (numbers as indicated) were picked for each and then purified plasmids were digested by restriction enzyme (Sbf1/Afe1 or Afe1/Mlu1), respectively.
Source publication
ZIKV has emerged as a significant human pathogene for the severe neurological complications, including Guillain-Barré(GBS) syndrome in adults and a variety of fetal abnormalities such as microcephaly. A stable and efficient infectious clone of Brazilian ZIKV isolate is required to study pathogenesis of epidemic ZIKV and virus evolution impact on it...
Contexts in source publication
Context 1
... autochthonous transmission strain to construct infectious clone. This strain (Genbank: KU321639.1) was isolated from a patient who received a blood transfusion from an asymptomatic donor at the time of donation in São Paulo state, Brazil, in 2015 36 . Due to incomplete sequence of its 3′UTR, we replaced its 3′UTR with another strain (KU527068.1) which was isolated from the brain of a fetus with microcephaly 37 . The ZIKV sequence was then synthesized artificially in two halves. As infectious clone backbone vector, we used a low-copy vector pFK which works efficiently for HCV infectious clone (Fig. 1a). The second half part (sequence 2) of synthetic ZIKV cDNA was easy to clone into pFK backbone, but we failed consistently to clone first half part (sequence 1) (Fig. 1b). We hypothesized that toxicity caused by putative bacterial promoters in ZIKV cDNA may be accounta- ble for this 38 . Therefore, the first half part of ZIKA cDNA was subjected to analyzed for the prediction of putative bacterial promoters by using the Neural Network promoter program (http://www.fruitfly.org/seq_tools/pro- moter.html). As expected, 8 putative bacterial promoters with scores higher than 0.9 were found in ZIKA genome sequences 1-3000nt (cDNA) ( Table ...
Context 2
... autochthonous transmission strain to construct infectious clone. This strain (Genbank: KU321639.1) was isolated from a patient who received a blood transfusion from an asymptomatic donor at the time of donation in São Paulo state, Brazil, in 2015 36 . Due to incomplete sequence of its 3′UTR, we replaced its 3′UTR with another strain (KU527068.1) which was isolated from the brain of a fetus with microcephaly 37 . The ZIKV sequence was then synthesized artificially in two halves. As infectious clone backbone vector, we used a low-copy vector pFK which works efficiently for HCV infectious clone (Fig. 1a). The second half part (sequence 2) of synthetic ZIKV cDNA was easy to clone into pFK backbone, but we failed consistently to clone first half part (sequence 1) (Fig. 1b). We hypothesized that toxicity caused by putative bacterial promoters in ZIKV cDNA may be accounta- ble for this 38 . Therefore, the first half part of ZIKA cDNA was subjected to analyzed for the prediction of putative bacterial promoters by using the Neural Network promoter program (http://www.fruitfly.org/seq_tools/pro- moter.html). As expected, 8 putative bacterial promoters with scores higher than 0.9 were found in ZIKA genome sequences 1-3000nt (cDNA) ( Table ...
Similar publications
Background
Virus genome sequences, generated in ever-higher volumes, can provide new scientific insights and inform our responses to epidemics and outbreaks. To facilitate interpretation, such data must be organised and processed within scalable computing resources that encapsulate virology expertise. GLUE (Genes Linked by Underlying Evolution) is...
Introduction: Traditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gen...
Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared f...
Negative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well...
Aquaculture is the fastest growing industry worldwide. Aquatic diseases have had enormous economic and environmental impacts in the recent past and the emergence of new aquatic pathogens, particularly viruses, poses a continuous threat. Nevertheless, little is known about the diversity, abundance and evolution of fish viruses. We used a meta-transc...
Citations
... The E-V763M mutation significantly increases neurovirulence and the viral load in the brains of newborn CD-1 mice, increases transplacental transmission, and significantly increases their mortality [42]. The NS5-M2634V mutation does not have a significant effect on replication in various cell cultures as well as on pathogenesis and virulence in mice; nevertheless, this mutation is fixed in all modern Asian strains [59]. The prM-E143K mutation, characteristic of African strains, increases the cytopathic effect and the titers of intracellular and extracellular virions; allows much better attachment to the cell membrane and penetration into human cell lines TE617, SF268, and HMC3; and ensures the release of the virus [44]. ...
The Zika virus (ZIKV) is a widespread mosquito-borne pathogen. Phylogenetically, two lineages of ZIKV are distinguished: African and Asian–American. The latter became the cause of the 2015–2016 pandemic, with severe consequences for newborns. In West African countries, the African lineage was found, but there is evidence of the emergence of the Asian–American lineage in Cape Verde and Angola. This highlights the need to not only monitor ZIKV but also sequence the isolates.
In this article, we present a case report of Zika fever in a pregnant woman from Guinea identified in 2018. Viral RNA was detected through qRT-PCR in a serum sample. In addition, the seroconversion of anti-Zika IgM and IgG antibodies was detected in repeated blood samples. Subsequently, the virus was isolated from the C6/36 cell line. The detected ZIKV belonged to the African lineage, the Nigerian sublineage. The strains with the closest sequences were isolated from mosquitoes in Senegal in 2011 and 2015. In addition, we conducted the serological screening of 116 blood samples collected from patients presenting to the hospital of Faranah with fevers during the period 2018–2021. As a result, it was found that IgM-positive patients were identified each year and that the seroprevalence varied between 5.6% and 17.1%.
... The E-V763M mutation significantly increases neurovirulence and the viral load in the brains of newborn CD-1 mice, increases transplacental transmission, and significantly increases their mortality [42]. The NS5-M2634V mutation does not have a significant effect on replication in various cell cultures as well as on pathogenesis and virulence in mice; nevertheless, this mutation is fixed in all modern Asian strains [60]. The prM-E143K mutation, characteristic of African strains, increases the cytopathic effect, the titers of intracellular, and extracellular virions; allows much better attachment to the cell membrane and penetration into human cell lines TE617, SF268, and HMC3; and ensures the release of the virus [44]. ...
Zika virus (ZIKV) is a widespread mosquito-borne pathogen. Phylogenetically, two lineages of the ZIKV are distinguished: African and Asian-American. The latter became the cause of the 2015-2016 pandemic with severe defeat to newborns. In West African countries the African lineage has been found, but there is evidence of the emergence of Asian-American lineage in Cape Verde and Angola. This highlights the need not only to monitor the ZIKV, but also to sequence the isolates. In this article, we present a case report of Zika fever in a pregnant woman from Guinea, identified in 2018. Viral RNA was detected by qRT-PCR in serum sample. In addition, seroconversion of anti-Zika IgM and IgG antibodies was detected in repeated blood samples. Subsequently, the virus was isolated in C6/36 cell line. The detected ZIKV belonged to the African lineage, the Nigerian sublineage. The strains with the closest sequences were isolated from mosquitoes in Senegal in 2011 and 2015. In addition, we conducted serological screening of 116 blood samples collected from patients presenting to the hospital of Faranah with fevers during the period 2018-2021. As a result, it was found that IgM-positive patients occurred each year, seroprevalence varied between 5.6% and 17.1%.
... It could be hypothesized that the M2634V substitution in the American strains is responsible for the increased outbreak potential of the mutant virus in the Americas. However, recent studies have shown that the insignificant M2634V mutation in the NS5 protein enhances viral replication and transmission potential, indicating that the M2634V substitution is not responsible for enhancing the ZIKV epidemic potential 39,40 . Here, we found that the ZIKV Thai strains isolated in 2019-2020 were SVM, similar to most previously isolated Thai and other Southeast Asian strains. ...
A large national outbreak of chikungunya virus (CHIKV) was recently reported in Thailand. While dengue virus (DENV) infection tends to occur year-round with an upsurge in the rainy season, Zika virus (ZIKV) also circulates in the country. The overlap in the distribution of these viruses increased the probability of co-infections during the heightened CHIKV activity. By examining 1806 patient serum samples submitted for CHIKV diagnostics from October 2018-February 2020 (511 CHIKV-negatives and 1295 CHIKV-positives), we used real-time reverse transcription-polymerase chain reaction to identify DENV and ZIKV individually. A total of 29 ZIKV and 36 DENV single-infections were identified. Interestingly, 13 co-infection cases were observed, of which 8 were CHIKV/DENV, 3 were CHIKV/ZIKV, and 2 were DENV/ZIKV. There were six DENV genotypes (13 DENV-1 genotype I, 10 DENV-2 Asian I, 10 DENV-2 Cosmopolitan, 6 DENV-3 genotype I, 2 DENV-3 genotype III, and 5 DENV-4 genotype I). Additionally, ZIKV strains identified in this study either clustered with strains previously circulating in Thailand and Singapore, or with strains previously reported in China, French Polynesia, and the Americas. Our findings reveal the co-infection and genetic diversity patterns of mosquito-borne viruses circulating in Thailand.
... Among these phenotypic behaviors that are ascribable to those mutations, there is a remarkable point to be made that is closely linked to the persistence of ZIKV in MRT such as the NS1 protein which decreases the phosphorylation of TBK1, and IRF3 and suppress the induction of IFN-β [27]. In contrast, the substitutions in the PrM [28] and E [29] proteins are related to neurovirulence, and apparently, the NS5 mutation does not alter the viral replication [30]. Additionally, in this study, a mutation was found in the capsid protein mutation, C: D107E ( Figure S1), in the MH179341.1 strain, however, this substitution is not relevant, nor does it affect the virus assembly as the amino acids that have been replaced have similar properties [31]. ...
Zika virus (ZIKV), a flavivirus that is mainly transmitted by A. aegypti and A. albopictus and sexual transmission, has been documented and described. The ZIKV RNA detection in the semen of vasectomized men indicates that accessory glands such as the prostate could be a site of virus replication. In this study, we characterized the ZIKV infection, evaluated the antiviral profile, and demonstrated the AXL and TIM-1 expression on the PC3 prostate cell line. It was also determined that PC3 cells are susceptible and permissive to ZIKV infection without altering the cell viability or causing a cytopathic effect. The antiviral profile suggests that the PC3 cells modulate the antiviral response through the suppressor molecule expression, SOCS-1, during a ZIKV infection.
... The data showed that the GFP levels and Renilla luciferase activities in the Go 6983-or bisindolylmaleimide I-treated cells were comparable to those in the DMSO-treated cells ( Fig. 4B and C), suggesting that the protein translation of plasmids was not affected by PKC inhibitors. Next, to test whether the PKC inhibitors specifically block the protein translation of SARS-CoV-2, we utilized a Zika virus (ZIKV) replicon (27). The data showed that Go 6983 and bisindolylmaleimide I barely affected the replication levels of the ZIKV replicon at both 6 and 24 h posttransfection (Fig. 4D), indicating that PKC inhibitors do not broadly inhibit viral translation and replication. ...
Infection by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has posed a severe threat to global public health. The current study revealed that several inhibitors of protein kinases C (PKCs) possess protective activity against SARS-CoV-2 infection. Four pan-PKC inhibitors, Go 6983, bisindolylmaleimide I, enzastaurin, and sotrastaurin, reduced the replication of a SARS-CoV-2 replicon in both BHK-21 and Huh7 cells. A PKCδ-specific inhibitor, rottlerin, was also effective in reducing viral infection. The PKC inhibitors acted at an early step of SARS-CoV-2 infection. Finally, PKC inhibitors blocked the replication of wild-type SARS-CoV-2 in ACE2-expressing A549 cells. Our work highlights the importance of the PKC signaling pathway in infection by SARS-CoV-2 and provides evidence that PKC-specific inhibitors are potential therapeutic agents against SARS-CoV-2.
IMPORTANCE There is an urgent need for effective therapeutic drugs to control the pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We found that several inhibitors of protein kinases C (PKCs) dramatically decrease the replication of SARS-CoV-2 in cultured cells. These PKC inhibitors interfere with an early step of viral infection. Therefore, the rapid and prominent antiviral effect of PKC inhibitors underscores that they are promising antiviral agents and suggests that PKCs are important host factors involved in infection by SARS-CoV-2.
... A previous study did not find a significant effect of NS5-M114V change on ZIKV replication in cells as well as pathogenesis and virulence in interferon receptor alpha/beta and gamma deficient (Ifnar-α/β/γ -/-) AG6 mice [28]. However, the impact of this mutation on virus virulence in the Ifnar-α/β -/mice that remain competent in IFNγ response and transmission potential in Ae. aegypti was not investigated. ...
... To determine the infection rate of each strain at 7 dpi (A) and 14 dpi (C), the number of respective ZIKV-positive bodies, legs and wings, and saliva expectorates were measured and expressed as percentages for comparison. To determine the efficiency of virus replication, dissemination and transmission potential, viral titers from ZIKV-positive bodies, legs and wings, and saliva expectorates at 7 dpi (B) and 14 dpi (D) were quantified and expressed as log 10 corresponding M114 mutant virus [28]. Our findings agreed with this study such that mutations at position 114 (either M➔V or V➔M) have no impact on virus replication in cells. ...
... These mutations therefore were implied to contribute to emergence of ZIKV in the Pacific Islands and Americas. On the other hand, Shan et al [16] reported insignificant impacts of NS5-M114V mutation (acquired during virus migration to Americas) on ZIKV neurovirulence using CD1 mice and Zhao et al [28] also didn't find significant effects of NS5-M114V mutation on various virus properties as discussed above. Additionally, despite NS5-M114V mutation not being present in Asian ZIKV isolates circulating during 2015-2016, large outbreaks like the one in Singapore were still recorded [24,47]. ...
During 2015–2016, outbreaks of Zika virus (ZIKV) occurred in Southeast Asia and the Americas. Most ZIKV infections in humans are asymptomatic, while clinical manifestation is usually a self-limiting febrile disease with maculopapular rash. However, ZIKV is capable of inducing a range of severe neurological complications collectively described as congenital Zika syndrome (CZS). Notably, the scale and magnitude of outbreaks in Southeast Asia were significantly smaller compared to those in the Americas. Sequence comparison between epidemic-associated ZIKV strains from Southeast Asia with those from the Americas revealed a methionine to valine substitution at residue position 114 of the NS5 protein (NS5-M114V) in all the American isolates. Using an American isolate of ZIKV (Natal), we investigated the impact of NS5-M114V mutation on virus replication in cells, virulence in interferon (IFN) α/β receptor knockout ( Ifnar -/- ) mice, as well as replication and transmission potential in Aedes aegypti mosquitoes. We demonstrated that NS5-M114V mutation had insignificant effect on ZIKV replication efficiency in cells, its ability to degrade STAT2, and virulence in vivo , albeit viremia was slightly prolonged in mice. Furthermore, NS5-M114V mutation decreased mosquito infection and dissemination rates and had no effect on virus secretion into the saliva. Taken together, our findings support the notion that NS5-M114V mutation is unlikely to be a major determinant for virus replication and transmission potential.
... In our study, the MEME method detected this position as being under positive selection, likely due to the occurrence of mutations in the first and second codon positions that lead to three alternative alleles at low frequency (<5 per cent) (Supplementary Table S5). M/T2634V was found in all the sequences from the American outbreak (and was not present in French Polynesia) (as found in Pettersson et al. 2016;Liu, Shi, and Qin 2019), but no evidence of altered pathogenesis in mice was found for this mutation (Zhao et al. 2018). ...
The Zika virus (ZIKV) disease caused a public health emergency of international concern that started in February 2016. The overall number of ZIKV-related cases increased until November 2016, after which it declined sharply. While the evaluation of the potential risk and impact of future arbovirus epidemics remains challenging, intensified surveillance efforts along with a scale-up of ZIKV whole-genome sequencing provide an opportunity to understand the patterns of genetic diversity, evolution, and spread of ZIKV. However, a classification system that reflects the true extent of ZIKV genetic variation is lacking. Our objective was to characterize ZIKV genetic diversity and phylodynamics, identify genomic footprints of differentiation patterns, and propose a dynamic classification system that reflects its divergence levels. We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within- and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters. Two main genetic clusters were defined, ZA and ZB, which correspond to the already characterized 'African' and 'Asian' genotypes, respectively. Within ZB, two subgroups, ZB.1 and ZB.2, represent the Asiatic and the American (and Oceania) lineages, respectively. ZB.1 is further subdivided into ZB.1.0 (a basal Malaysia sequence sampled in the 1960s and a recent Indian sequence), ZB.1.1 (South-Eastern Asia, Southern Asia, and Micronesia sequences), and ZB.1.2 (very similar sequences from the outbreak in Singapore). ZB.2 is subdivided into ZB.2.0 (basal American sequences and the sequences from French Polynesia, the putative origin of South America introduction), ZB.2.1 (Central America), and ZB.2.2 (Caribbean and North America). This classification system does not use geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve analyses of ZIKV genomic variation and its association with pathogenicity and serve as a starting point for the public health surveillance and response to on-going and future epidemics and to outbreaks that lead to the emergence of new variants.
... For complete details on the use and execution of this protocol, please refer to Zeng et al. (2020) BEFORE YOU BEGIN ZIKV preparation from an infectious clone Timing: 8 days 1. Making ZIKV infectious clone plasmid a. ZIKV infectious clone containing a T7 promoter (Zhao et al., 2018) ( Figure 1A) is an ultralowcopy plasmid because it is cyclized through ZIKV genome sequence combined with Ampicillin resistant sequence. Maxiprep of the plasmid is necessary due to the large usage of the 3. ...
Zika virus (ZIKV) is linked to congenital defects including microcephaly. An infection model that can recapitulate most microcephaly-related phenotypes is crucial for understanding ZIKV pathogenesis. Here, we present a protocol to generate ZIKV from an infectious clone through a reverse genetic system and subsequently perform embryonic brain infection with the rescued ZIKV in pregnant mice. We optimized several aspects of the procedures including virus rescue and in utero injection. This protocol facilitates reproducible investigation of virus-induced cortical development defects.
For complete details on the use and execution of this protocol, please refer to Zeng et al. (2020)
... damage in mice 30,31 . A report suggests that an amino acid substitution that emerged after the ZVD epidemic in Brazil does not affect the replication and virulence of ZIKV 26,32 . Thus, the relationship between genetic variation in ZIKV and its replication capacity and pathogenicity remains controversial. ...
Zika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes.
... Infectious clones can be constructed in three types of systems. The first is the in vitro transcription system [23][24][25], where a full-length DNA clone is used as a template to transcribe viral mRNA molecules using T7 or SP6 RNA polymerase. The purified viral RNA is then used to transfect cells via electrotransfection or other methods to produce rescued viruses. ...
Background
Zika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP.
Methods
A PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP. ZIKA-EGFP virus was rescued by transfection of pBAC-ZIKA-EGFP into 293T cells. The characterization of ZIKA-EGFP virus was determined by qPCR, plaque assay, CCK-8, and Western blot.
Results
Rescued ZIKA-EGFP virus exhibited stable replication for at least five generations in tissue culture. ZIKA-EGFP can effectively infect C6/36, SH-SY5Y and Vero cells, and cause cytopathic effects on SH-SY5Y and Vero cells. The inhibition of ZIKA-EGFP by NF-κB inhibitor, caffeic acid phenethyl ester was observed by fluorescence microscopy.
Conclusion
Our results suggested that Zika virus infectious clone with an EGFP marker retained it infectivity as wide-type Zika virus which could be used for drugs screening.
