Ruey-Yi Chang - Academia.edu (original) (raw)

Papers by Ruey-Yi Chang

Research paper thumbnail of Japanese Encephalitis Virus Genotype Replacement, Taiwan, 2009–2010

Emerging Infectious Diseases, 2011

Genotype I of Japanese encephalitis virus fi rst appeared in Taiwan in 2008. Phylogenetic analysi... more Genotype I of Japanese encephalitis virus fi rst appeared in Taiwan in 2008. Phylogenetic analysis of 37 viruses from pig farms in 2009-2010 classifi ed these viruses into 2 unique subclusters of genotype I viruses and suggested multiple introductions and swift replacement of genotype III by genotype I virus in Taiwan.

Research paper thumbnail of A cis-acting function for the coronavirus leader in defective interfering RNA replication

Journal of Virology, 1994

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5&#39... more To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of termi...

Research paper thumbnail of Leader-Body Junction Sequence of the Viral Subgenomic mRNAs of Porcine Reproductive and Respiratory Syndrome Virus Isolated in Taiwan

Journal of Veterinary Medical Science, 2002

Research paper thumbnail of A phylogenetically conserved hairpin-type 39 untranslated region pseudoknot functions in coronavirus RNA replication

This article cites 67 articles, 37 of which can be accessed free

Research paper thumbnail of An Insight into Cancer and Anticancer Drugs

Acta Scientific Medical Sciences

Cancer is one of the most common and widespread diseases in the human population and has been rep... more Cancer is one of the most common and widespread diseases in the human population and has been reported to be the second major cause of death globally. The World Health Organisation states that about 200 different types of cancers have been identified worldwide, each of which requires unique approaches for treatment. It also records that 1 in every 6 deaths in the world are due to cancer which clearly evidences for the inevitable necessity for the control and cure of cancer. Unlike normal cells, cancer cells undergo uncontrollable cell division and each division leads to multiple mutations in a continuous fashion. There are various strategies towards targeting cancerous cells. With the advent of conventional therapies, various chemical derivatives are being used as anticancer drugs. This article reviews on the various approaches put forth in the fight against cancer through cancer therapies and drug designing by identifying potent targets. Here we will be discussing about the history of cancer and its advances towards development of anticancer drugs from tip to toe.

Research paper thumbnail of The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

PLOS ONE

Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported t... more Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported that sfRNA results from stalling of host 5'-to-3' exoribonuclease XRN1 at the highly structured RNA of the 3' untranslated region (UTR). Although XRN1 digestion of a 3'-terminal 800-nt RNA could stall at a position to generate the sfRNA in vitro, we found that knocking out XRN1 had no effect on the accumulation of sfRNA in Japanese encephalitis virus (JEV) infected cells. Mutagenesis studies revealed that the stemloop II (SLII) at the 3' UTR is required for the accumulation of sfRNA. According to the results of an in vitro RNAdependent RNA polymerase (RdRp) assay, the (-)10431-10566 RNA fragment, containing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Taken together, our results indicate that the JEV sfRNA could be transcribed initially and then be trimmed by XRN1 or other unidentified exoribonucleases.

Research paper thumbnail of Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan

Emerging infectious diseases, Nov 1, 2017

The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigat... more The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigated differences in the virulence of GI and GIII JEV by calculating asymptomatic ratios based on serologic studies during GI- and GIII-JEV endemic periods. The results suggested equal virulence of GI and GIII JEV among humans.

Research paper thumbnail of Article URL

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted P... more This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3 ' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

Research paper thumbnail of A Phylogenetically Conserved Hairpin-Type 39 Untranslated Region Pseudoknot Functions in Coronavirus RNA Replication

Secondary and tertiary structures in the 3* untranslated region (UTR) of plus-strand RNA viruses ... more Secondary and tertiary structures in the 3* untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3* UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required

Research paper thumbnail of Leader-mRNA junction sequences are unique for each subgenomic mRNA species in the bovine coronavirus and remain so throughout persistent infection

Virology, 1993

The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To e... more The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3' end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junctio...

Research paper thumbnail of Role of subgenomic minus-strand RNA in coronavirus replication

Archives of virology. Supplementum, 1994

Coronavirus subgenomic minus-strand RNAs (negative-strand copies of the 3' coterminal subgeno... more Coronavirus subgenomic minus-strand RNAs (negative-strand copies of the 3' coterminal subgenomic mRNAs) probably function in mRNA amplification by serving as templates for transcription from internal (intergenic) promoters, rather than by faithful (full-length) mRNA replication.

Research paper thumbnail of cis Requirement for N-specific protein sequence in bovine coronavirus defective interfering RNA replication

Journal of virology, 1996

A naturally occurring 2.2-kb defective interfering (DI) RNA of the bovine coronavirus, structural... more A naturally occurring 2.2-kb defective interfering (DI) RNA of the bovine coronavirus, structurally a simple fusion of the genomic termini, contains a single contiguous open reading frame (ORF) or 1.7 kb composed of the 5'-terminal 288 nucleotides of polymerase gene 1a and all 1,344 nucleotides of the nucleocapsid protein (N) gene. The ORF must remain open throughout most of its sequence for replication to occur. To determine the qualitative importance of the N portion of the chimeric ORF in DI RNA replication, transcripts of mutated reporter-containing constructs were tested for replication in helper virus-infected cells. It was determined that the N ORF could not be replaced by the naturally occurring internal I protein ORF, accomplished by deleting the first base in the N start codon which leads to a +1 frameshift, nor could it be replaced by the chloramphenicol acetyltransferase ORF. Furthermore, 3'-terminal truncations of the N gene leaving less than 85% of its total le...

Research paper thumbnail of A phylogenetically conserved hairpin-type 3' untranslated region pseudoknot functions in coronavirus RNA replication

Journal of virology, 1999

Secondary and tertiary structures in the 3' untranslated region (UTR) of plus-strand RNA viru... more Secondary and tertiary structures in the 3' untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3' UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required for the replication of a defective interfering RNA genome. The pseudoknot is phylogenetically conserved among coronaviruses both in location and in shape but only partially in nucleotide sequence, and evolutionary covariation of bases to maintain G. U pairings indicates that it functions in the plus strand. RNase probing of synthetic transcripts provided additional evidence of its tertiary structure and also identified the possible existence of two conformational states. These results indicate that the 3' UTR pseudoknot is involved in coronavirus RNA replication and...

Research paper thumbnail of Defective interfering RNAs of Japanese encephalitis virus found in mosquito cells and correlation with persistent infection

Virus Research, 2007

Defective interfering (DI) RNAs are deletion mutants of viral genomes that are known in many case... more Defective interfering (DI) RNAs are deletion mutants of viral genomes that are known in many cases to contribute to persistent infection and modification of viral pathogenesis. Cell type also plays a critical role in the establishment of viral persistence. In this study we have identified for the first time the generation of DI RNAs of Japanese encephalitis virus in C6/36 mosquito cells. A persistent infection was established by replacing growth medium on surviving cells and continued cell passaging. Persistent infection was demonstrated by a continual release of infectious virus, fluorescent antibody staining, and Northern analysis. A population of DI RNAs of approximately 8.2-9.7 kb, not detectable in acutely infected cells, became apparent in the persistently infected cells by 25 days postinfection. Sequence analyses revealed a population of DI RNAs that contained in-frame deletions of 1.3-2.8 kb covering the region of the E gene and some flanking C or prM and NS1 gene sequences. Transcripts from one cDNA clone of a DI RNA replicated in uninfected mosquito cells as demonstrated by RT-PCR. DI RNA-containing virions in supernatant fluids from persistently infected mosquito cells could be used to establish persistent infection in BHK-21 cells. The correlation of DI RNA presence with cell survival suggests that DI RNAs are contributing mechanistically to the establishment of persistent infection in both the mosquito and mammalian cells.

Research paper thumbnail of Full genome analysis of a novel type II feline coronavirus NTU156

Virus Genes, 2013

Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated... more Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.

Research paper thumbnail of Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

Virology Journal, 2011

Background: Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese ence... more Background: Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese encephalitis virus (JEV) are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA) which is collinear with the highly conserved regions of the 3'-UTR in JEVinfected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results: Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome) reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Transacting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions: Our results indicate that sfRNA modulates viral translation and replication in trans.

Research paper thumbnail of DnaJ homolog Hdj2 Facilitates Japanese Encephalitis Virus Replication

Virology Journal, 2011

Background Japanese encephalitis virus (JEV) is a member of the mosquito-borne Flaviviridae famil... more Background Japanese encephalitis virus (JEV) is a member of the mosquito-borne Flaviviridae family of viruses that causes human encephalitis. Upon infection of a new host, replication of viral RNA involves not only the viral RNA-dependent RNA polymerase (RdRp), but also host proteins. Host factors involved in JEV replication are not well characterized. Results We identified Hdj2, a heat-shock protein 40 (Hsp40)/DnaJ homolog, from a mouse brain cDNA library interacting with JEV nonstructural protein 5 (NS5) encoding viral RdRp using yeast two-hybrid system. Specific interaction of Hdj2 with NS5 was confirmed by coimmunoprecipitation and colocalization in JEV-infected cells. Overexpression of Hdj2 in JEV-infected cells led to an increase of RNA synthesis, and the virus titer was elevated approximately 4.5- to 10-fold. Knocking down of Hdj2 by siRNA reduced the virus production significantly. Conclusions We conclude that Hdj2 directly associates with JEV NS5 and facilitates viral repli...

Research paper thumbnail of Tandem Placement of a Coronavirus Promoter Results in Enhanced mRNA Synthesis from the Downstream-Most Initiation Site

Virology, 1996

Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-... more Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3-most on the genome) promoter. These results show that enhancement of subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.

Research paper thumbnail of Japanese encephalitis virus non-coding RNA inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3

Veterinary Microbiology, 2013

Noncoding RNA (ncRNA) plays a critical role in modulating a broad range of diseases. All arthropo... more Noncoding RNA (ncRNA) plays a critical role in modulating a broad range of diseases. All arthropod-borne flaviviruses produce short fragment ncRNA (sfRNA) collinear with highly conserved regions of the 3'-untranslated region (UTR) in the viral genome. We show that the molar ratio of sfRNA to genomic RNA in Japanese encephalitis virus (JEV) persistently infected cells is greater than that in acutely infected cells, indicating an sfRNA role in establishing persistent infection. Transfecting excess quantities of sfRNA into JEV-infected cells reduced interferon-β (IFN-β) promoter activity by 57% and IFN-β mRNA levels by 52%, compared to mock-transfected cells. Transfection of sfRNA into JEV-infected cells also reduced phosphorylation of interferon regulatory factor-3 (IRF-3), the IFN-β upstream regulator, and blocked roughly 30% of IRF-3 nuclear localization. Furthermore, JEV-infected sfRNA transfected cells produced 23% less IFN-β-stimulated apoptosis than mock-transfected groups did. Taken together, these results suggest that sfRNA plays a role against host-cell antiviral responses, prevents cells from undergoing apoptosis, and thus contributes to viral persistence.

Research paper thumbnail of Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Molecular Genetics and Genomics, 2001

Dierent regions of RF DNA from the ®lamentous bacteriophage /Lf were cloned in Escherichia coli v... more Dierent regions of RF DNA from the ®lamentous bacteriophage /Lf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-speci®c integration. Mutations in himA, which codes for the a-subunit of the Integration Host Factor, does not aect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the /Lf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-speci®c integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-speci®c recombination, including the 6-bp central region, and 8/ 11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp /Lf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-speci®c integration is neither speci®ed by the 4445-bp Xc17 chromosomal region nor encoded by the /Lf genome.

Research paper thumbnail of Japanese Encephalitis Virus Genotype Replacement, Taiwan, 2009–2010

Emerging Infectious Diseases, 2011

Genotype I of Japanese encephalitis virus fi rst appeared in Taiwan in 2008. Phylogenetic analysi... more Genotype I of Japanese encephalitis virus fi rst appeared in Taiwan in 2008. Phylogenetic analysis of 37 viruses from pig farms in 2009-2010 classifi ed these viruses into 2 unique subclusters of genotype I viruses and suggested multiple introductions and swift replacement of genotype III by genotype I virus in Taiwan.

Research paper thumbnail of A cis-acting function for the coronavirus leader in defective interfering RNA replication

Journal of Virology, 1994

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5&#39... more To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of termi...

Research paper thumbnail of Leader-Body Junction Sequence of the Viral Subgenomic mRNAs of Porcine Reproductive and Respiratory Syndrome Virus Isolated in Taiwan

Journal of Veterinary Medical Science, 2002

Research paper thumbnail of A phylogenetically conserved hairpin-type 39 untranslated region pseudoknot functions in coronavirus RNA replication

This article cites 67 articles, 37 of which can be accessed free

Research paper thumbnail of An Insight into Cancer and Anticancer Drugs

Acta Scientific Medical Sciences

Cancer is one of the most common and widespread diseases in the human population and has been rep... more Cancer is one of the most common and widespread diseases in the human population and has been reported to be the second major cause of death globally. The World Health Organisation states that about 200 different types of cancers have been identified worldwide, each of which requires unique approaches for treatment. It also records that 1 in every 6 deaths in the world are due to cancer which clearly evidences for the inevitable necessity for the control and cure of cancer. Unlike normal cells, cancer cells undergo uncontrollable cell division and each division leads to multiple mutations in a continuous fashion. There are various strategies towards targeting cancerous cells. With the advent of conventional therapies, various chemical derivatives are being used as anticancer drugs. This article reviews on the various approaches put forth in the fight against cancer through cancer therapies and drug designing by identifying potent targets. Here we will be discussing about the history of cancer and its advances towards development of anticancer drugs from tip to toe.

Research paper thumbnail of The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

PLOS ONE

Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported t... more Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported that sfRNA results from stalling of host 5'-to-3' exoribonuclease XRN1 at the highly structured RNA of the 3' untranslated region (UTR). Although XRN1 digestion of a 3'-terminal 800-nt RNA could stall at a position to generate the sfRNA in vitro, we found that knocking out XRN1 had no effect on the accumulation of sfRNA in Japanese encephalitis virus (JEV) infected cells. Mutagenesis studies revealed that the stemloop II (SLII) at the 3' UTR is required for the accumulation of sfRNA. According to the results of an in vitro RNAdependent RNA polymerase (RdRp) assay, the (-)10431-10566 RNA fragment, containing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Taken together, our results indicate that the JEV sfRNA could be transcribed initially and then be trimmed by XRN1 or other unidentified exoribonucleases.

Research paper thumbnail of Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan

Emerging infectious diseases, Nov 1, 2017

The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigat... more The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigated differences in the virulence of GI and GIII JEV by calculating asymptomatic ratios based on serologic studies during GI- and GIII-JEV endemic periods. The results suggested equal virulence of GI and GIII JEV among humans.

Research paper thumbnail of Article URL

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted P... more This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3 ' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

Research paper thumbnail of A Phylogenetically Conserved Hairpin-Type 39 Untranslated Region Pseudoknot Functions in Coronavirus RNA Replication

Secondary and tertiary structures in the 3* untranslated region (UTR) of plus-strand RNA viruses ... more Secondary and tertiary structures in the 3* untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3* UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required

Research paper thumbnail of Leader-mRNA junction sequences are unique for each subgenomic mRNA species in the bovine coronavirus and remain so throughout persistent infection

Virology, 1993

The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To e... more The common leader sequence on bovine coronavirus subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3' end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junctio...

Research paper thumbnail of Role of subgenomic minus-strand RNA in coronavirus replication

Archives of virology. Supplementum, 1994

Coronavirus subgenomic minus-strand RNAs (negative-strand copies of the 3' coterminal subgeno... more Coronavirus subgenomic minus-strand RNAs (negative-strand copies of the 3' coterminal subgenomic mRNAs) probably function in mRNA amplification by serving as templates for transcription from internal (intergenic) promoters, rather than by faithful (full-length) mRNA replication.

Research paper thumbnail of cis Requirement for N-specific protein sequence in bovine coronavirus defective interfering RNA replication

Journal of virology, 1996

A naturally occurring 2.2-kb defective interfering (DI) RNA of the bovine coronavirus, structural... more A naturally occurring 2.2-kb defective interfering (DI) RNA of the bovine coronavirus, structurally a simple fusion of the genomic termini, contains a single contiguous open reading frame (ORF) or 1.7 kb composed of the 5'-terminal 288 nucleotides of polymerase gene 1a and all 1,344 nucleotides of the nucleocapsid protein (N) gene. The ORF must remain open throughout most of its sequence for replication to occur. To determine the qualitative importance of the N portion of the chimeric ORF in DI RNA replication, transcripts of mutated reporter-containing constructs were tested for replication in helper virus-infected cells. It was determined that the N ORF could not be replaced by the naturally occurring internal I protein ORF, accomplished by deleting the first base in the N start codon which leads to a +1 frameshift, nor could it be replaced by the chloramphenicol acetyltransferase ORF. Furthermore, 3'-terminal truncations of the N gene leaving less than 85% of its total le...

Research paper thumbnail of A phylogenetically conserved hairpin-type 3' untranslated region pseudoknot functions in coronavirus RNA replication

Journal of virology, 1999

Secondary and tertiary structures in the 3' untranslated region (UTR) of plus-strand RNA viru... more Secondary and tertiary structures in the 3' untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3' UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required for the replication of a defective interfering RNA genome. The pseudoknot is phylogenetically conserved among coronaviruses both in location and in shape but only partially in nucleotide sequence, and evolutionary covariation of bases to maintain G. U pairings indicates that it functions in the plus strand. RNase probing of synthetic transcripts provided additional evidence of its tertiary structure and also identified the possible existence of two conformational states. These results indicate that the 3' UTR pseudoknot is involved in coronavirus RNA replication and...

Research paper thumbnail of Defective interfering RNAs of Japanese encephalitis virus found in mosquito cells and correlation with persistent infection

Virus Research, 2007

Defective interfering (DI) RNAs are deletion mutants of viral genomes that are known in many case... more Defective interfering (DI) RNAs are deletion mutants of viral genomes that are known in many cases to contribute to persistent infection and modification of viral pathogenesis. Cell type also plays a critical role in the establishment of viral persistence. In this study we have identified for the first time the generation of DI RNAs of Japanese encephalitis virus in C6/36 mosquito cells. A persistent infection was established by replacing growth medium on surviving cells and continued cell passaging. Persistent infection was demonstrated by a continual release of infectious virus, fluorescent antibody staining, and Northern analysis. A population of DI RNAs of approximately 8.2-9.7 kb, not detectable in acutely infected cells, became apparent in the persistently infected cells by 25 days postinfection. Sequence analyses revealed a population of DI RNAs that contained in-frame deletions of 1.3-2.8 kb covering the region of the E gene and some flanking C or prM and NS1 gene sequences. Transcripts from one cDNA clone of a DI RNA replicated in uninfected mosquito cells as demonstrated by RT-PCR. DI RNA-containing virions in supernatant fluids from persistently infected mosquito cells could be used to establish persistent infection in BHK-21 cells. The correlation of DI RNA presence with cell survival suggests that DI RNAs are contributing mechanistically to the establishment of persistent infection in both the mosquito and mammalian cells.

Research paper thumbnail of Full genome analysis of a novel type II feline coronavirus NTU156

Virus Genes, 2013

Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated... more Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.

Research paper thumbnail of Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

Virology Journal, 2011

Background: Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese ence... more Background: Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese encephalitis virus (JEV) are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA) which is collinear with the highly conserved regions of the 3'-UTR in JEVinfected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results: Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome) reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Transacting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions: Our results indicate that sfRNA modulates viral translation and replication in trans.

Research paper thumbnail of DnaJ homolog Hdj2 Facilitates Japanese Encephalitis Virus Replication

Virology Journal, 2011

Background Japanese encephalitis virus (JEV) is a member of the mosquito-borne Flaviviridae famil... more Background Japanese encephalitis virus (JEV) is a member of the mosquito-borne Flaviviridae family of viruses that causes human encephalitis. Upon infection of a new host, replication of viral RNA involves not only the viral RNA-dependent RNA polymerase (RdRp), but also host proteins. Host factors involved in JEV replication are not well characterized. Results We identified Hdj2, a heat-shock protein 40 (Hsp40)/DnaJ homolog, from a mouse brain cDNA library interacting with JEV nonstructural protein 5 (NS5) encoding viral RdRp using yeast two-hybrid system. Specific interaction of Hdj2 with NS5 was confirmed by coimmunoprecipitation and colocalization in JEV-infected cells. Overexpression of Hdj2 in JEV-infected cells led to an increase of RNA synthesis, and the virus titer was elevated approximately 4.5- to 10-fold. Knocking down of Hdj2 by siRNA reduced the virus production significantly. Conclusions We conclude that Hdj2 directly associates with JEV NS5 and facilitates viral repli...

Research paper thumbnail of Tandem Placement of a Coronavirus Promoter Results in Enhanced mRNA Synthesis from the Downstream-Most Initiation Site

Virology, 1996

Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-... more Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3-most on the genome) promoter. These results show that enhancement of subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.

Research paper thumbnail of Japanese encephalitis virus non-coding RNA inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3

Veterinary Microbiology, 2013

Noncoding RNA (ncRNA) plays a critical role in modulating a broad range of diseases. All arthropo... more Noncoding RNA (ncRNA) plays a critical role in modulating a broad range of diseases. All arthropod-borne flaviviruses produce short fragment ncRNA (sfRNA) collinear with highly conserved regions of the 3'-untranslated region (UTR) in the viral genome. We show that the molar ratio of sfRNA to genomic RNA in Japanese encephalitis virus (JEV) persistently infected cells is greater than that in acutely infected cells, indicating an sfRNA role in establishing persistent infection. Transfecting excess quantities of sfRNA into JEV-infected cells reduced interferon-β (IFN-β) promoter activity by 57% and IFN-β mRNA levels by 52%, compared to mock-transfected cells. Transfection of sfRNA into JEV-infected cells also reduced phosphorylation of interferon regulatory factor-3 (IRF-3), the IFN-β upstream regulator, and blocked roughly 30% of IRF-3 nuclear localization. Furthermore, JEV-infected sfRNA transfected cells produced 23% less IFN-β-stimulated apoptosis than mock-transfected groups did. Taken together, these results suggest that sfRNA plays a role against host-cell antiviral responses, prevents cells from undergoing apoptosis, and thus contributes to viral persistence.

Research paper thumbnail of Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Molecular Genetics and Genomics, 2001

Dierent regions of RF DNA from the ®lamentous bacteriophage /Lf were cloned in Escherichia coli v... more Dierent regions of RF DNA from the ®lamentous bacteriophage /Lf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-speci®c integration. Mutations in himA, which codes for the a-subunit of the Integration Host Factor, does not aect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the /Lf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-speci®c integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-speci®c recombination, including the 6-bp central region, and 8/ 11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp /Lf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-speci®c integration is neither speci®ed by the 4445-bp Xc17 chromosomal region nor encoded by the /Lf genome.