Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression

Journal of Neuroimmunology

Woolley

et al. 2017

Self Cite

282

222

Zika virus (ZIKV), a mosquito-borne positive-stranded RNA virus of the family Flaviviridae (genus Flavivirus), is now causing an unprecedented large-scale outbreak in the Americas. Historically, ZIKV spread eastward from equatorial Africa and Asia to the Pacific Islands during the late 2000s to early 2010s, invaded the Caribbean and Central and South America in 2015, and reached North America in 2016. Although ZIKV infection generally causes no symptoms or only a mild self-limiting illness, it has recently been linked to a rising number of severe neurological diseases, including microcephaly and Guillain-Barré syndrome. Because of the continuous geographic expansion of both the virus and its mosquito vectors, ZIKV poses a serious threat to public health around the globe. However, there are no vaccines or antiviral therapies available against this pathogen. This review summarizes a fast-growing body of literature on the history, epidemiology, transmission, and clinical presentation of ZIKV and highlights the urgent need for the development of efficient control strategies for this emerging pathogen.

Section: Virologymentioning

confidence: 99%

Zika virus: History, epidemiology, transmission, and clinical presentation

Song

Journal of Neuroimmunology

Woolley

et al. 2017

Self Cite

282

222

“…Thus, viral replication can be initiated by the introduction of a cDNA-derived genome-length RNA molecule into a susceptible host cell. The availability of an infectious JEV cDNA clone, when combined with recombinant DNA technology, has increased our understanding of various aspects of the viral life cycle at the molecular level, such as gene expression 73,79 and genome replication. 63,64 Also, a full-length JEV cDNA clone has proven to be a valuable tool for the development of antiviral vaccines 28 and gene delivery vectors.…”

Section: Discussionmentioning

confidence: 99%

“…Optional: Examine RNA-electroporated cells at 18-20 hr post-transfection for JEV protein expression by immunofluorescence assays 27,73 (Figure 6B), and harvest the supernatants from the RNA-electroporated cells at 22 and 40 hr post-transfection for virus titration by plaque assays 27,63 (Figure 6C).…”

mentioning

confidence: 99%

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Song

Kim

et al. 2015

JoVE

Self Cite

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

“…The ORF in the JEV genomic RNA encodes a polyprotein precursor of ~3432 amino acids, which is cleaved into at least 10 distinct products [ 141 , 142 ], i.e., three structural (capsid, C; premembrane, prM; and envelope, E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins ( Figure 1 B,C). In flaviviruses, the site-specific proteolysis of the polyprotein is catalyzed co- and post-translationally by a set of four different proteases: (i) the host signal peptidase responsible for cleaving at the C-prM, prM-E, E-NS1, and NS4A-NS4B junctions within the lumen of the endoplasmic reticulum (ER) [ 143 , 144 , 145 , 146 , 147 , 148 ]; (ii) the two-component viral protease NS3 + NS2B [ 149 ] required for cleaving at the NS2A-NS2B, NS2B-NS3, NS3-NS4A, and NS4B-NS5 junctions, as well as at internal sites within the C and NS4A proteins on the cytoplasmic face of the ER membrane [ 143 , 144 , 150 , 151 , 152 , 153 , 154 , 155 ]; (iii) the host furin or furin-like protease mediating the final cleavage of prM to M in the trans -Golgi network [ 156 ]; and (iv) an unknown host protease capable of cleaving at the NS1-NS2A junction [ 157 , 158 , 159 ].…”

Section: Jev Is a Small Enveloped Positive-strand Rna Virusmentioning

confidence: 99%

“…Once inside the endosome, the viral E glycoprotein undergoes low pH-induced conformational changes [ 207 , 208 , 209 , 210 , 211 , 212 , 213 ], triggering the fusion of viral and host endosomal membranes [ 214 , 215 , 216 , 217 , 218 , 219 , 220 , 221 ]. Following membrane fusion, the genomic RNA is released into the cytoplasm, where it is translated into two precursor polyproteins (with or without a ribosomal frameshifting at the beginning of NS2A-coding region) that are cleaved to yield three structural (C, prM, and E) and at least seven nonstructural (NS1 to NS5) proteins, along with NS1′ [ 141 , 142 ].…”

Section: Jev Is a Small Enveloped Positive-strand Rna Virusmentioning

confidence: 99%

Early Events in Japanese Encephalitis Virus Infection: Viral Entry

Lee

2018

Pathogens

Self Cite

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic flavivirus, is an enveloped positive-strand RNA virus that can cause a spectrum of clinical manifestations, ranging from mild febrile illness to severe neuroinvasive disease. Today, several killed and live vaccines are available in different parts of the globe for use in humans to prevent JEV-induced diseases, yet no antivirals are available to treat JEV-associated diseases. Despite the progress made in vaccine research and development, JEV is still a major public health problem in southern, eastern, and southeastern Asia, as well as northern Oceania, with the potential to become an emerging global pathogen. In viral replication, the entry of JEV into the cell is the first step in a cascade of complex interactions between the virus and target cells that is required for the initiation, dissemination, and maintenance of infection. Because this step determines cell/tissue tropism and pathogenesis, it is a promising target for antiviral therapy. JEV entry is mediated by the viral glycoprotein E, which binds virions to the cell surface (attachment), delivers them to endosomes (endocytosis), and catalyzes the fusion between the viral and endosomal membranes (membrane fusion), followed by the release of the viral genome into the cytoplasm (uncoating). In this multistep process, a collection of host factors are involved. In this review, we summarize the current knowledge on the viral and cellular components involved in JEV entry into host cells, with an emphasis on the initial virus-host cell interactions on the cell surface.