scholarly journals Human Rotavirus Reverse Genetics Systems to Study Viral Replication and Pathogenesis

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1791
Author(s):  
Satoshi Komoto ◽  
Saori Fukuda ◽  
Takayuki Murata ◽  
Koki Taniguchi
Keyword(s):  
Viral Replication ◽  
Rational Design ◽  
Reverse Genetics ◽  
Helper Virus ◽  
Review Article ◽  
Historical Background ◽  
Proper Understanding ◽  
Human Rotavirus ◽  
Research Questions ◽  
Infants And Young Children

Human rotaviruses (HuRVAs) are highly important causes of acute gastroenteritis in infants and young children worldwide. A lack of reliable and reproducible reverse genetics systems for HuRVAs has limited a proper understanding of HuRVA biology and also the rational design of live-attenuated vaccines. Since the development of the first reverse genetics system for RVAs (partially plasmid-based reverse genetics system) in 2006, there have been many efforts with the goal of generating infectious recombinant HuRVAs entirely from cloned cDNAs. However, the establishment of a HuRVA reverse genetics system was very challenging until 2019. This review article provides an overview of the historical background of the recent development of long-awaited HuRVA reverse genetics systems, beginning with the generation of recombinant human-simian reassortant RVAs with the aid of a helper virus in 2006 and the generation of recombinant animal (simian) RVAs in a helper virus-free manner in 2017, and culminating in the generation of recombinant HuRVAs entirely from plasmid cDNAs in 2019. Notably, the original HuRVA reverse genetics system has already been optimized to increase the efficiency of virus generation. Although the application of HuRVA reverse genetics systems has only just been initiated, these technologies will help to answer HuRVA research questions regarding viral replication and pathogenicity that could not be addressed before, and to develop next-generation vaccines and intestine-specific rotaviral vectors.

scholarly journals Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 201 ◽  
Author(s):  
Alexander Falkenhagen ◽  
Corinna Patzina-Mehling ◽  
Ashish K. Gadicherla ◽  
Amy Strydom ◽  
Hester G. O’Neill ◽  
...  
Keyword(s):  
Cell Culture ◽  
Disease Burden ◽  
Reverse Genetics ◽  
Sub Saharan Africa ◽  
Rotavirus A ◽  
Virus Particles ◽  
Rotavirus Vaccines ◽  
Human Rotavirus ◽  
Sub Saharan ◽  
Infants And Young Children

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.

scholarly journals Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

2010 ◽  
Vol 84 (13) ◽  
pp. 6711-6719 ◽  
Author(s):  
Cécile Troupin ◽  
Axelle Dehée ◽  
Aurélie Schnuriger ◽  
Patrice Vende ◽  
Didier Poncet ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Helper Virus ◽  
Wild Type ◽  
Human Rotavirus ◽  
Protein Functions ◽  
Group A Rotaviruses ◽  
Group A

ABSTRACT Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

First Insights on Peruvian Luxury Consumers

2018 ◽  
Vol 9 (8) ◽  
pp. 699-712
Author(s):  
Anne-Flore Maman Larraufie ◽  
Keyword(s):  
Emerging Markets ◽  
Cultural Dimensions ◽  
Historical Background ◽  
Two Stage ◽  
Secondary Sources ◽  
Strong Potential ◽  
Analytic Process ◽  
Emerging Country ◽  
Research Questions ◽  
Practical Recommendations

Peru is an emerging country showing strong potential for future luxury developments. It already holds luxury regular consumers, mainly in the Lima capital. However, it is currently approached in a standardized process by luxury firms, following what is done in other emerging markets for luxury. To be efficient, it is necessary to get more knowledge about Peruvian consumers. This is what this article aims at. After reviewing the historical background of the country along with its cultural dimensions, we present results from a two-stage analytic process based on data collected from secondary sources and interviews with consumers. We derive from that practical recommendations for luxury managers and propose some research questions and hypotheses to be further explored and tested.

scholarly journals The use of ceramics as an aesthetic element in Durban architecture (1914-2012)

2013 ◽  
Author(s):  
◽  
Raksha Padaruth
Keyword(s):  
Historical Background ◽  
Art Practice ◽  
Local Identity ◽  
Architectural Elements ◽  
Architectural Element ◽  
Wide Range ◽  
Research Questions ◽  
Contemporary Practice ◽  
Insight Into

This paper documents and evaluates the use of ceramics as an aesthetic architectural element in Durban from 1914-2012 with special reference to James Hall (1916-2006), Andrew Walford (b.1942) and Jane du Rand (b.1969). These artists were selected because their work demonstrates a wide range of the use of decorative tiles and mosaics as aesthetic elements in Durban architecture over a period of more than fifty years. Reference is made to the historical use of tiles and mosaics as aesthetic architectural elements in Durban from 1914-1955 in order to provide a context to an investigation and evaluation of the contribution of Hall, Walford and du Rand to the use of tiles and mosaics as an aesthetic architectural element in Durban. The paper begins by highlighting the importance of this study, discusses the role of ceramic architectural adornment and defines terminology for the purpose of this research. In addition an explanation of the research methodology used, research questions and literature review is provided. The study is contextualised through an overview of the historical background of the use of ceramics (tiles and mosaics) as an aesthetic element in architecture. The importance of the use of ceramic elements in relation to architecture, as well as the different techniques and methods of production, are highlighted and related to contemporary practice. The overview provides insight into how the use of ceramic elements in the past has influenced the approach of contemporary practice. My contribution to the use of mosaics as an aesthetic architectural element in Durban and my art practice, in the form of an installation titled passage is discussed and evaluated. The paper concludes by noting that the historical use of tiles and mosaics as aesthetic elements in architecture persists in contemporary art practice. However, the methods of tiled mosaic production and tiled mosaic techniques have been revolutionised extensively. It is evident that, the use of ceramics as an aesthetic element in Durban architecture reflects, both a strong European design influence and a distinctive local identity.

scholarly journals Socio-Spatial Research on Logistics and Its Impact on Urban Environment: A Review

2021 ◽  
Vol 16 (1) ◽  
pp. 61-69
Author(s):  
José A. López-Sánchez ◽  
Beltrán Roca
Keyword(s):  
Spatial Analysis ◽  
Urban Environment ◽  
Urban Space ◽  
Economic Activity ◽  
Applied Research ◽  
Review Article ◽  
Transport Activity ◽  
Academic Literature ◽  
The Usa ◽  
Research Questions

This review article attempts to respond to the following research questions: how has evolved socio-spatial analysis on logistics, which are the most studied types of this economic activity, and the major impacts of logistics on urban space. This article draws on a systemized bibliometric analysis to identify the main tendencies in logistics' spatial study. It identifies four clusters of literature that put interest on different subtopics and approaches. The review reveals the current hegemony of applied research that focuses on sustainability, streamlining, and technology, mainly from the USA and China, despite research on globalization and industry. In fact, concerning the urban space, the most vivid academic discussion revolves around the location of warehousing and transport activity within cities. Finally, the article highlights the lack of critical perspectives on logistics and socio-spatial conflicts generated by logistics extension in mainstream academic literature. The analysis concludes that socio-spatial disputes related to logistics remain understudied and, consequently, further research should be conducted on this field.

scholarly journals Assembly and Maintenance of Sarcomere Thin Filaments and Associated Diseases

2020 ◽  
Vol 21 (2) ◽  
pp. 542 ◽  
Author(s):  
Kendal Prill ◽  
John F. Dawson
Keyword(s):  
Thin Filament ◽  
Reverse Genetics ◽  
Medical Intervention ◽  
Model Organisms ◽  
Skeletal Muscle Function ◽  
Thin Filaments ◽  
Physiological Processes ◽  
Open Research ◽  
Research Questions ◽  
Sarcomere Assembly

Sarcomere assembly and maintenance are essential physiological processes required for cardiac and skeletal muscle function and organism mobility. Over decades of research, components of the sarcomere and factors involved in the formation and maintenance of this contractile unit have been identified. Although we have a general understanding of sarcomere assembly and maintenance, much less is known about the development of the thin filaments and associated factors within the sarcomere. In the last decade, advancements in medical intervention and genome sequencing have uncovered patients with novel mutations in sarcomere thin filaments. Pairing this sequencing with reverse genetics and the ability to generate patient avatars in model organisms has begun to deepen our understanding of sarcomere thin filament development. In this review, we provide a summary of recent findings regarding sarcomere assembly, maintenance, and disease with respect to thin filaments, building on the previous knowledge in the field. We highlight debated and unknown areas within these processes to clearly define open research questions.

scholarly journals Surveillance of Human Rotavirus in Wuhan, China (2011–2019): Predominance of G9P[8] and Emergence of G12

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 810
Author(s):  
Xuan Zhou ◽  
Yuan-Hong Wang ◽  
Bei-Bei Pang ◽  
Nan Chen ◽  
Nobumichi Kobayashi
Keyword(s):  
Etiologic Agent ◽  
Genetic Characteristics ◽  
Predominant Genotype ◽  
Rotavirus A ◽  
Epidemic Season ◽  
Human Rotavirus ◽  
Stool Specimens ◽  
Virus Strains ◽  
Nsp3 Gene ◽  
Infants And Young Children

Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children worldwide. To learn the shift of genotypes and genetic characteristics of Rotavirus A (RVA) causing diarrhea in children and adults, a hospital-based surveillance of rotavirus was conducted in Wuhan, China from June 2011 through May 2019, and representative virus strains were phylogenetically analyzed. Among a total of 6733 stool specimens collected from both children and adults with acute gastroenteritis, RVA was detected in 25.5% (1125/4409) and 12.3% (285/2324) of specimens, respectively. G9P[8] was the most common genotype (74.5%), followed by G1P[8] (8.7%), G2P[4] (8.4%), and G3P[8] (7.3%), with G9P[8] increasing rapidly during the study period. The predominant genotype shifted from G1P[8] to G9P[8] in 2012–2013 epidemic season. G12P[6] strain RVA/Human-wt/CHN/Z2761/2019/G12P[6] was detected in April 2019 and assigned to G12-P[6]-I1-R1-C1-M1-A1-N1-T2-E1-H1 genotypes. Phylogenetic analysis revealed that VP7, VP4, VP6, VP3, NSP1, NSP2, and NSP5 genes of Z2761 clustered closely with those of Korean G12P[6] strain CAU_214, showing high nucleotide identities (98.0–98.8%). The NSP3 gene of Z2761 was closely related to those of G2P[4] and G12P[6] rotaviruses in Asia. All the eleven gene segments of Z2761 kept distance from those of cocirculating G9P[8], G1P[8], and G3P[8] strains detected in Wuhan during this study period. This is the first identification of G12 rotavirus in China. It is deduced that Z2761 is a reassortant having DS-1-like NSP3 gene in the background of G12P[6] rotavirus genetically close to CAU_214.

scholarly journals Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

2015 ◽  
Vol 89 (9) ◽  
pp. 4907-4917 ◽  
Author(s):  
Anna M. Mielech ◽  
Xufang Deng ◽  
Yafang Chen ◽  
Eveline Kindler ◽  
Dorthea L. Wheeler ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protease Activity ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Viral Pathogenesis ◽  
Wild Type ◽  
Viral Protease ◽  
Novel Approach ◽  
Protease Stability

ABSTRACTUbiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

scholarly journals Restriction of Viral Replication by Mutation of the Influenza Virus Matrix Protein

2002 ◽  
Vol 76 (24) ◽  
pp. 13055-13061 ◽  
Author(s):  
Teresa Liu ◽  
Zhiping Ye
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Zinc Finger ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Reverse Genetics ◽  
Rna Binding ◽  
Viral Assembly ◽  
Zinc Finger Motif

ABSTRACT The matrix protein (M1) of influenza virus plays an essential role in viral assembly and has a variety of functions, including association with influenza virus ribonucleoprotein (RNP). Our previous studies show that the association of M1 with viral RNA and nucleoprotein not only promotes formation of helical RNP but also is required for export of RNP from the nucleus during viral replication. The RNA-binding domains of M1 have been mapped to two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105, which is also involved in RNP-binding activity. To further understand the role of the RNP-binding domain of M1 in viral assembly and replication, mutations in the coding sequences of RKLKR and the zinc finger motif of M1 were constructed using a PCR technique and introduced into wild-type influenza virus by reverse genetics. Altering the zinc finger motif of M1 only slightly affected viral growth. Substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR resulted in a lethal mutation. These results indicate that the RKLKR domain of M1 protein plays an important role in viral replication.

scholarly journals Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Satoshi Komoto ◽  
Yuta Kanai ◽  
Saori Fukuda ◽  
Masanori Kugita ◽  
Takahiro Kawagishi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Growth Curves ◽  
Single Step ◽  
Replication Cycle ◽  
Nonstructural Proteins ◽  
Virus Growth ◽  
Severe Diarrhea

ABSTRACT The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

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