scholarly journals Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
L. Mueller ◽  
P. M. Hauser ◽  
F. Gauye ◽  
G. Greub
Keyword(s):  
Dihydrofolate Reductase ◽  
Expression System ◽  
Open Reading Frames ◽  
Dna Viruses ◽  
Content Type ◽  
The Family ◽  
Nucleocytoplasmic Large Dna Viruses ◽  
First Time ◽  
Reading Frames

ABSTRACT Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV.

scholarly journals Genome Sequence of the Broad-Host-Range Pseudomonas Phage Φ-S1

2012 ◽  
Vol 86 (18) ◽  
pp. 10239-10239 ◽  
Author(s):  
Sanna Sillankorva ◽  
Andrew M. Kropinski ◽  
Joana Azeredo
Keyword(s):  
Host Range ◽  
Genome Sequence ◽  
Open Reading Frames ◽  
Broad Host Range ◽  
Content Type ◽  
Double Stranded Dna ◽  
The Family ◽  
Reading Frames

The broad-host-range lyticPseudomonasphage Φ-S1 possess a 40,192 bp double-stranded DNA (dsDNA) genome of 47 open reading frames (ORFs) and belongs to the familyPodoviridae, subfamilyAutographivirinae, genusT7likevirus.

scholarly journals First Report of Macrolide Resistance Geneerm(T) Harbored by a Novel Small Plasmid from Erysipelothrix rhusiopathiae

2015 ◽  
Vol 59 (4) ◽  
pp. 2462-2465 ◽  
Author(s):  
Chang-Wen Xu ◽  
An-Yun Zhang ◽  
Chun-Mei Yang ◽  
Yun Pan ◽  
Zhong-Bin Guan ◽  
...  
Keyword(s):  
Low Frequency ◽  
Macrolide Resistance ◽  
Open Reading Frames ◽  
The Novel ◽  
Erysipelothrix Rhusiopathiae ◽  
Small Plasmid ◽  
Content Type ◽  
Macrolide Resistance Gene ◽  
First Time ◽  
Reading Frames

ABSTRACTThe macrolide resistance geneerm(T) was identified for the first time in a porcineErysipelothrix rhusiopathiaeisolate from swine in China. The novel 3,749-bp small plasmid pER29, which carrieserm(T), had a G+C content of 31% and four distinct open reading frames. The presence of pER29 increased by at least 128-fold the MICs of clindamycin and erythromycin forE. rhusiopathiae. The fitness cost of pER29 could be responsible for the low frequency oferm(T) inE. rhusiopathiae.

scholarly journals Phage ϕC2 Mediates Transduction of Tn6215, Encoding Erythromycin Resistance, between Clostridium difficile Strains

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Shan Goh ◽  
Haitham Hussain ◽  
Barbara J. Chang ◽  
Warren Emmett ◽  
Thomas V. Riley ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Genetic Manipulation ◽  
Recipient Strain ◽  
Open Reading Frames ◽  
Human Pathogen ◽  
Erythromycin Resistance ◽  
Content Type ◽  
Filter Mating ◽  
First Time ◽  
Reading Frames

ABSTRACTIn this work, we show thatClostridium difficilephage ϕC2 transduceserm(B), which confers erythromycin resistance, from a donor to a recipient strain at a frequency of 10−6per PFU. The transductants were lysogenic for ϕC2 and contained theerm(B) gene in a novel transposon, Tn6215. This element is 13,008 bp in length and contains 17 putative open reading frames (ORFs). It could also be transferred at a lower frequency by filter mating.IMPORTANCEClostridium difficileis a major human pathogen that causes diarrhea that can be persistent and difficult to resolve using antibiotics.C. difficileis potentially zoonotic and has been detected in animals, food, and environmental samples.C. difficilegenomes contain large portions of horizontally acquired genetic elements. The conjugative elements have been reasonably well studied, but transduction has not yet been demonstrated. Here, we show for the first time transduction as a mechanism for the transfer of a novel genetic element inC. difficile. Transduction may also be a useful tool for the genetic manipulation ofC. difficile.

scholarly journals Rescue of Two Recombinant Canine Distemper Viruses That Separately Express Dabie Bandavirus Gn and Gc in Vitro

2021 ◽  
Author(s):  
Fuxiao Liu ◽  
Jiahui Lin ◽  
Qianqian Wang ◽  
Hu Shan
Keyword(s):  
Reverse Genetics ◽  
Open Reading Frames ◽  
Canine Distemper ◽  
Significant Difference ◽  
The Family ◽  
Potential Vaccine ◽  
Severe Fever ◽  
Reading Frames

Abstract Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis with a high mortality rate in humans. Additionally, dogs are frequently reported to be infected with this disease. There has been no commercially available vaccine for humans and animals as yet. The SFTS is caused by Dabie bandavirus (DBV), formerly known as SFTS virus. The DBV is now classified into the genus Bandavirus in the family Phenuiviridae. DBV Gn and Gc can induce specific immune responses in vivo. In this study, we used reverse genetics to construct two recombinant canine distemper viruses (rCDV), rCDV-Gn and -Gc, which could express Dabie bandavirus Gn and Gc in vitro, respectively. Two foreign sequences, Gn and Gc open reading frames, were genetically stable during twenty serial viral passages in cells. Growth curve of the rCDV-Gc basically coincided with that of a wild-type CDV, but showed a significant difference from that of the rCDV-Gn. The rCDV-Gn and -Gc were derived from a common parental CDV, the virulence-attenuating QN strain. Therefore, if proven to be efficient in resisting both canine distemper and SFTS in dogs, either or both recombinant CDVs would be potential vaccine candidates.

scholarly journals Generation of a Stable Plasmid forIn VitroandIn VivoStudies of Staphylococcus Species

2016 ◽  
Vol 82 (23) ◽  
pp. 6859-6869 ◽  
Author(s):  
Christina N. Krute ◽  
Kelsey L. Krausz ◽  
Mary A. Markiewicz ◽  
Jason A. Joyner ◽  
Srijana Pokhrel ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Antibiotic Usage ◽  
Open Reading Frames ◽  
Antibiotic Selection ◽  
Content Type ◽  
Plasmid Maintenance ◽  
Genetic Tools ◽  
Reading Frames

ABSTRACTA major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as duringin vivomodels of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid inStaphylococcus aureusUSA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools forStaphylococcusspecies. LAC-p01 was genetically manipulated into anEscherichia coli-S. aureusshuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (dsoandsso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use inS. aureusUSA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable inStaphylococcus epidermidis. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model ofS. aureussystemic infection and successfully complemented anhlamutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during bothin vitroandin vivoexperiments are powerful new tools for studies ofStaphylococcus.IMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation duringin vivostudies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from anS. aureusUSA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generationsin vitroandin vivowithout antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies ofStaphylococcusspecies.

scholarly journals Whole-Genome Sequence of Oryctes rhinoceros Nudivirus from Riau Province, Indonesia

2021 ◽  
Vol 10 (48) ◽  
Author(s):  
Yudistira Wahyu Kurnia ◽  
Zulfikar Achmad Tanjung ◽  
Condro Utomo ◽  
Mohammad Naim ◽  
Elizabeth Caroline Situmorang ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Open Reading Frames ◽  
Whole Genome Sequence ◽  
Dna Virus ◽  
Content Type ◽  
Oryctes Rhinoceros ◽  
Double Stranded Dna ◽  
The Family ◽  
Total Dna ◽  
Reading Frames

A double-stranded DNA virus, Oryctes rhinoceros nudivirus (OrNV), was detected in the total DNA of diseased larvae of O. rhinoceros in Riau Province, Indonesia. The complete genome sequence was 124,926 bp long and encodes 123 open reading frames (ORFs). This strain belongs to the family Nudiviridae and was designated LiboV.

scholarly journals The Novel Macrolide-Lincosamide-Streptogramin B Resistance Geneerm(44) Is Associated with a Prophage in Staphylococcus xylosus

2014 ◽  
Vol 58 (10) ◽  
pp. 6133-6138 ◽  
Author(s):  
Juliette R. K. Wipf ◽  
Sybille Schwendener ◽  
Vincent Perreten
Keyword(s):  
Bovine Mastitis ◽  
Open Reading Frames ◽  
Regulatory Sequence ◽  
The Novel ◽  
Direct Repeats ◽  
Content Type ◽  
Staphylococcus Xylosus ◽  
The Family ◽  
Methylase Gene ◽  
Reading Frames

ABSTRACTA novel erythromycin ribosome methylase gene,erm(44), that confers resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics was identified by whole-genome sequencing of the chromosome ofStaphylococcus xylosusisolated from bovine mastitis milk. Theerm(44) gene is preceded by a regulatory sequence that encodes two leader peptides responsible for the inducible expression of the methylase gene, as demonstrated by cloning inStaphylococcus aureus. Theerm(44) gene is located on a 53-kb putative prophage designated ΦJW4341-pro. The 56 predicted open reading frames of ΦJW4341-pro are structurally organized into the five functional modules found in members of the familySiphoviridae. ΦJW4341-pro is site-specifically integrated into theS. xylosuschromosome, where it is flanked by two perfect 19-bp direct repeats, and exhibits the ability to circularize. The presence oferm(44) in three additionalS. xylosusstrains suggests that this putative prophage has the potential to disseminate MLSBresistance.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Genomic Sequence of Rhesus Cytomegalovirus 180.92: Insights into the Coding Potential of Rhesus Cytomegalovirus

2006 ◽  
Vol 80 (8) ◽  
pp. 4179-4182 ◽  
Author(s):  
Pierre Rivailler ◽  
Amitinder Kaur ◽  
R. Paul Johnson ◽  
Fred Wang
Keyword(s):  
Genomic Sequence ◽  
Open Reading Frames ◽  
Rhesus Cytomegalovirus ◽  
Human Cmv ◽  
Coding Capacity ◽  
Coding Potential ◽  
Reading Frames ◽  
Genetic Rearrangements

ABSTRACT A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.

scholarly journals Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Shili Yang ◽  
Lijuan Zhao ◽  
Ruipeng Ma ◽  
Wei Fang ◽  
Jia Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Agrotis Segetum ◽  
Foreign Protein ◽  
Content Type ◽  
Occlusion Bodies ◽  
Foreign Proteins ◽  
Novel Strategy

ABSTRACT The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo. The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity. IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

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