scholarly journals Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
James Weger-Lucarelli ◽  
Nisha K. Duggal ◽  
Kristen Bullard-Feibelman ◽  
Milena Veselinovic ◽  
Hannah Romo ◽  
...  
Keyword(s):  
Puerto Rico ◽  
Zika Virus ◽  
New World ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Infectious Clone ◽  
Clinical Manifestations ◽  
Primary Isolate ◽  
Content Type

ABSTRACT Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

scholarly journals Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Zhong-Yu Liu ◽  
Jiu-Yang Yu ◽  
Xing-Yao Huang ◽  
Hang Fan ◽  
Xiao-Feng Li ◽  
...  
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Neurological Complications ◽  
Cdna Clones ◽  
Content Type ◽  
Infectious Clones ◽  
Global Concern ◽  
Rna Elements ◽  
Self Splicing

ABSTRACT Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5′-SLA and 5′-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

scholarly journals Erratum for Weger-Lucarelli et al., “Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone”

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
James Weger-Lucarelli ◽  
Nisha K. Duggal ◽  
Kristen Bullard-Feibelman ◽  
Milena Veselinovic ◽  
Hannah Romo ◽  
...  
Keyword(s):  
Zika Virus ◽  
Recombinant Virus ◽  
New World ◽  
Infectious Clone

scholarly journals Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

10.3791/55857 ◽  
2017 ◽  
Author(s):  
James Weger-Lucarelli ◽  
Nisha K. Duggal ◽  
Aaron C. Brault ◽  
Brian J. Geiss ◽  
Gregory D. Ebel
Keyword(s):  
Zika Virus ◽  
Recombinant Virus ◽  
New World ◽  
Infectious Clone

scholarly journals CD4 T Cell Antigens from Staphylococcus aureus Newman Strain Identified following Immunization with Heat-Killed Bacteria

2012 ◽  
Vol 19 (4) ◽  
pp. 477-489 ◽  
Author(s):  
Paulraj K. Lawrence ◽  
Bachra Rokbi ◽  
Nadège Arnaud-Barbe ◽  
Eric L. Sutten ◽  
Junzo Norimine ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
T Cell ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Protein A ◽  
Cd4 T Cell ◽  
Content Type ◽  
Intramammary Infections ◽  
T Cell Antigens ◽  
Ifn Γ

ABSTRACTStaphylococcus aureusis a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistantS. aureus(MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens fromS. aureushas made it difficult to design effective vaccines. The goal of this study was to identifyS. aureusproteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from theS. aureusNewman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.

scholarly journals Establishment, Validation, and Application of a New World Primate Model of EnterotoxigenicEscherichia coliDisease for Vaccine Development

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
Julianne E. Rollenhagen ◽  
Franca Jones ◽  
Eric Hall ◽  
Ryan Maves ◽  
Gladys Nunez ◽  
...  
Keyword(s):  
New World ◽  
Vaccine Development ◽  
World Monkey ◽  
Dose Range ◽  
Cholera Toxin B Subunit ◽  
Primate Model ◽  
Content Type ◽  
Fecal Shedding ◽  
B Subunit ◽  
Series Of Experiments

ABSTRACTThe establishment of an animal model that closely approximates enterotoxigenicEscherichia coli(ETEC) disease in humans is critical for the development and evaluation of vaccines against this enteropathogen. Here, we evaluated the susceptibility ofAotus nancymaae, a New World monkey species, to ETEC infection. Animals were challenged orogastrically with 109to 1011CFU of the human pathogenic CFA/I+ETEC strain H10407 and examined for evidence of diarrhea and fecal shedding of bacteria. A clear dose-range effect was obtained, with diarrheal attack rates of 40% to 80%, validated in a follow-on study demonstrating an attack rate of 80% with 1011CFU of H10407 ETEC. To determine whether this model is an effective approach for assessing ETEC vaccine candidates, we used it to evaluate the ability of the donor strand-complemented CFA/I adhesin CfaE (dscCfaE) to protect against H10407 challenge. In a series of experiments, animals were intranasally vaccinated with dscCfaE alone, dscCfaE with either cholera toxin B-subunit (CTB) or heat-labile toxin (LTB), or phosphate-buffered saline (PBS) alone and then challenged with 1011CFU of H10407. Control animals vaccinated with PBS had attack rates of 70 to 90% on challenge. Vaccination with dscCfaE, or dscCfaE admixed with CTB or LTB, resulted in a reduction of attack rates, with vaccine efficacies of 66.7% (P = 0.02), 77.7% (P = 0.006), and 42.9% (P = 0.370) to 83.3% (P = 0.041), respectively. In conclusion, we have shown the H10407 ETEC challenge ofA. nancymaaeto be an effective, reproducible model of ETEC disease, and importantly, we have demonstrated that in this model, vaccination with the prototype vaccine candidate dscCfaE is protective against CF-homologous disease.

scholarly journals Molecular and epidemiological characterization of human adenoviruses infection among children with acute diarrhea in Shandong Province, China

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Deyu Huang ◽  
Zheng Wang ◽  
Guanyou Zhang ◽  
Lintao Sai
Keyword(s):  
Acute Diarrhea ◽  
Vaccine Development ◽  
Seasonal Pattern ◽  
Clinical Manifestations ◽  
Pcr Amplification ◽  
Shandong Province ◽  
Positive Rate ◽  
Epidemiological Characterization ◽  
Children Under 5

Abstract Background Human adenovirus (HAdV) had been recognized as one of the most common enteric viruses associated with acute diarrhea in children. The present study was carried out to demonstrate the molecular and epidemiological characterization of HAdV Infections among children in Shandong province in China between July 2017 and June 2018. Methods Fecal specimens were collected from children under 5 years old with acute diarrhea. DNA was extracted from the stool specimens and adenovirus DNA was detected by PCR amplification with specific primers. The amplification products were subjected to electrophoresis and visualized on a UV transilluminator. All positive RT-PCR amplification products were sequenced and the obtained sequences analyzed by MEGA (version 7.0). Demographic information and clinical manifestation data were also analyzed. Results In total, 656 fecal specimens were collected and the overall positive rate of HAdV was 7.47%. HAdV infections were detected in all age groups, in which children aged 13–24 months presented the highest positive rate. Seasonal pattern could be observed with a peak in December, January and February. Diarrhea, vomiting, dehydration and fever were the main clinical manifestations, in which vomiting was the most common accompanied symptom. By phylogenetic analysis, four species (A, B, C, and F) were detected and seven different serotypes were identified. HAdV-41 (48.98%, 24/49) was the most common serotype followed by HAdV-3 (18.37%, 9/49), HAdV-31 (14.29%, 7/49), HAdV-7 (8.16%, 4/49), HAdV-40 (4.08%, 2/49), HAdV-1 (4.08%, 2/49) and HAdV-2 (2.04%, 1/49). Conclusion This study indicated that HAdV infection was an important cause of acute diarrhea among children under 5 years old in Shandong province. The results will contribute to (a) increase understanding of the role of HAdV in diarrheal children and enhance identification of the predominant diarrhea pathogen for diagnosis; (b) avoid abuse of antibiotics; (c) monitor the change of prevalent HAdV serotypes and promote vaccine development and vaccination.

scholarly journals Complete Genome Sequences of Three Historically Important, Spatiotemporally Distinct, and Genetically Divergent Strains of Zika Virus: MR-766, P6-740, and PRVABC-59: TABLE 1

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Sang-Im Yun ◽  
Byung-Hak Song ◽  
Jordan C. Frank ◽  
Justin G. Julander ◽  
Irina A. Polejaeva ◽  
...  
Keyword(s):  
Puerto Rico ◽  
Zika Virus ◽  
Complete Genome ◽  
Reverse Genetics ◽  
Genomic Sequences ◽  
Genome Sequences ◽  
African Lineage ◽  
American Strain ◽  
Asian Lineage ◽  
Virus Strains

Here, we report the 10,807-nucleotide-long consensus RNA genome sequences of three spatiotemporally distinct and genetically divergent Zika virus strains, with the functionality of their genomic sequences substantiated by reverse genetics: MR-766 (African lineage, Uganda, 1947), P6-740 (Asian lineage, Malaysia, 1966), and PRVABC-59 (Asian lineage-derived American strain, Puerto Rico, 2015).

scholarly journals Development and characterization of infectious clones of two strains of Usutu virus

2020 ◽  
Author(s):  
Tyler Bates ◽  
Christina Chuong ◽  
Seth A. Hawks ◽  
Pallavi Rai ◽  
Rebecca M. Salgado ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Insect Cells ◽  
Rna Virus ◽  
Infectious Clone ◽  
Bird Species ◽  
Infection Transmission ◽  
Usutu Virus ◽  
Infectious Clones ◽  
Positive Sense

AbstractUsutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. In this report, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in both mammalian and insect cells. Additionally, we observed similar levels of replication and pathogenesis in two mouse models. This reverse genetics system will aid the scientific community in studying and developing USUV infection, transmission, diagnostics, and vaccines.

scholarly journals A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Konstantin A. Tsetsarkin ◽  
Heather Kenney ◽  
Rubing Chen ◽  
Guangping Liu ◽  
Hasmik Manukyan ◽  
...  
Keyword(s):  
Vero Cell ◽  
Cell Lines ◽  
Cdna Clone ◽  
Zika Virus ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Infectious Cdna Clone ◽  
Guillain Barre Syndrome ◽  
Perinatal Development ◽  
Guillain Barré

ABSTRACTAn arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics.IMPORTANCEThe availability of genetic tools and laboratory models determines the progress in understanding mechanisms of virus emergence and pathogenesis. Recent large-scale outbreaks of Zika virus (ZIKV) that were linked to complications during perinatal development and Guillain-Barré syndrome in adults emphasize the urgency for the development of a reverse-genetics system based on an epidemic ZIKV strain. Here, we report a stable infectious cDNA clone for ZIKV isolated during the 2015 epidemic in Brazil, as well as a Vero cell-adapted version of it, which will be used for virus-host interaction studies and vaccine development.

scholarly journals Construction of a Full-length Infectious Clone of Zika Virus Stably Expressing EGFP Marker in a Eukaryotic Expression System

2020 ◽  
Author(s):  
Jing Gao ◽  
Lingjuan Shi ◽  
Jiayi Chen ◽  
Weizhi Lu ◽  
Jingtai Cai ◽  
...  
Keyword(s):  
Zika Virus ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Expression System ◽  
Plaque Assay ◽  
Vero Cells ◽  
Full Length ◽  
Egfp Expression ◽  
Wild Type ◽  
Significant Difference

Abstract Background: Zika virus is among the most widely transmitted arboviruses in the world and closely associated with diseases, such as encephalitis, fetal microcephaly, and Guillain–Barré syndrome. The pathogenic mechanism of the virus has not been fully elucidated, and there are no vaccines or specific drugs targeting the virus. To address these issues, the application of reverse genetics is needed for viral reconstruction and reproduction.Methods: Polymerase chain reaction (PCR) was used to merge the full-length Zika virus genome, CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into a pBAC11 vector through recombination to produce recombinant pBAC-ZIKA-EGFP. The ZIKA–EGFP was rescued by transfection of 293T cells with pBAC-ZIKA-EGFP, and at 7-days post-transfection, the supernatant (P0 generation) was passed through a 0.45-μm membrane and used to infect Vero cells (to produce the P1 generation). Fluorescence-based quantitative PCR, 50% tissue culture infectious dose, and plaque assays were used to measure differences in replication ability and pathogenicity relative to the rescue virus (ZIKA–WT), the sequence of which is consistent with that of the wild-type Zika virus. Additionally, caffeic acid phenethyl ester (CAPE), a nuclear factor kappaB (NF-kB) inhibitor, was used to examine its effect on viral replication.Results: The results showed that ZIKA–EGFP could effectively infect Vero cells, SH-SY5Y cells and C6/36 cells, and cause cytopathic effects on them. ZIKA–EGFP exhibited stable replication and EGFP expression during cell passage for at least six generations, with no significant difference in replication ability relative to the ZIKA–WT. Fluorescent cell foci were observed in the plaque assay while the ZIKA–EGFP was in the absence of phage plaque formation. The inhibition of NF-kB inhibitor on ZIKA-EGFP was observed by fluorescence microscopy, which was consistent with the results of fluorescence quantitative PCR.Conclusions: We constructed an infectious clone of the full-length genome of Zika virus which could replicate with stable EGFP expression in eukaryotic cells during passage. The infectious clone, remaining main characteristics of wild type ZIKA virus could be appied on the studies of reverse genetics, drug screening and gene function of ZIKA virus.

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