Our Technophile
Akela G
Past Master’s student
Ms. Ghazawi defended her Master’s thesis entitled, “Fine mapping of sequences important for FIV RNA packaging and their mechanism of function” successfully in April 2005. Work from her thesis resulted in two publications (J. Virol., 2005; Microbes Infect., 2006). In addition, based on her contributions to different ongoing projects in the laboratory, she has been a co-author on several other publications from our laboratory. Akela pursued her PhD degree from University of Pecs, Hungary in 2012 and is currently working as a Medical Research Specialist at CMHS, UAE University.
Publications
2022
Pál, T.; Butt, A. B.; Ghazawi, A.; Thomsen, J.; Rizvi, T. A.; Sonnevend, Á
Early Years of Carbapenem-Resistant Enterobacterales Epidemic in Abu Dhabi Journal Article
In: Antibiotics (Basel), vol. 11, no. 10, 2022, ISSN: 2079-6382 (Print) 2079-6382.
@article{RN17,
title = {Early Years of Carbapenem-Resistant Enterobacterales Epidemic in Abu Dhabi},
author = {T. Pál and A. B. Butt and A. Ghazawi and J. Thomsen and T. A. Rizvi and Á Sonnevend},
doi = {10.3390/antibiotics11101435},
issn = {2079-6382 (Print) 2079-6382},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Antibiotics (Basel)},
volume = {11},
number = {10},
abstract = {Recent studies showed that the current endemic of carbapenem-resistant Enterobacterales (CRE) in the Emirate of Abu Dhabi is dominated by highly resistant Klebsiella pneumoniae clones ST14, ST231, and CC147, respectively. In the absence of continuous, molecular typing-based surveillance, it remained unknown whether they lately emerged and rapidly became dominant, or they had been present from the early years of the endemic. Therefore, antibiotic resistance, the presence of carbapenemase and 16S methylase genes, and the sequence types of CRE strains collected between 2009 and 2015 were compared with those collected between 2018 and 2019. It was found that members of these three clones, particularly those of the most prevalent ST14, started dominating already in the very early years of the CRE outbreak. Furthermore, while severely impacting the overall antibiotic resistance patterns, the effect of these clones was not exclusive: for example, increasing trends of colistin or decreasing rates of tigecycline resistance were also observed among nonclonal isolates. The gradually increasing prevalence of few major, currently dominating clones raises the possibility that timely, systematic, molecular typing-based surveillance could have provided tools to public health authorities for an early interference with the escalation of the local CRE epidemic.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent studies showed that the current endemic of carbapenem-resistant Enterobacterales (CRE) in the Emirate of Abu Dhabi is dominated by highly resistant Klebsiella pneumoniae clones ST14, ST231, and CC147, respectively. In the absence of continuous, molecular typing-based surveillance, it remained unknown whether they lately emerged and rapidly became dominant, or they had been present from the early years of the endemic. Therefore, antibiotic resistance, the presence of carbapenemase and 16S methylase genes, and the sequence types of CRE strains collected between 2009 and 2015 were compared with those collected between 2018 and 2019. It was found that members of these three clones, particularly those of the most prevalent ST14, started dominating already in the very early years of the CRE outbreak. Furthermore, while severely impacting the overall antibiotic resistance patterns, the effect of these clones was not exclusive: for example, increasing trends of colistin or decreasing rates of tigecycline resistance were also observed among nonclonal isolates. The gradually increasing prevalence of few major, currently dominating clones raises the possibility that timely, systematic, molecular typing-based surveillance could have provided tools to public health authorities for an early interference with the escalation of the local CRE epidemic.
Sonnevend, Á; Abdulrazzaq, N.; Ghazawi, A.; Thomsen, J.; Bharathan, G.; Makszin, L.; Rizvi, T. A.; Pál, T.
In: Int J Infect Dis, vol. 120, pp. 103-112, 2022, ISSN: 1201-9712.
@article{RN30,
title = {The first nationwide surveillance of carbapenem-resistant Enterobacterales in the United Arab Emirates - increased association of Klebsiella pneumoniae CC14 clone with Emirati patients},
author = {Á Sonnevend and N. Abdulrazzaq and A. Ghazawi and J. Thomsen and G. Bharathan and L. Makszin and T. A. Rizvi and T. Pál},
doi = {10.1016/j.ijid.2022.04.034},
issn = {1201-9712},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Int J Infect Dis},
volume = {120},
pages = {103-112},
abstract = {OBJECTIVES: To assess the current prevalence, distribution, and main clonal types of carbapenem-resistant Enterobacterales (CRE) in the United Arab Emirates. METHODS: A total of 504 CRE collected over a 9-month period in 15 hospitals were studied. Antibiotic susceptibility and the presence of common carbapenemase, 16S methylase, and mobile colistin resistance genes were assessed. Selected strains forming larger clusters by pulsed field gel electrophoresis were subjected to whole genome sequencing to identify their sequence types and core genome MLST. RESULTS: Strains expressing OXA and NDM type carbapenemases and 16S methylases were present in all major hospitals. Considerable interhospital differences were noticed, suggesting the role of specific clones. A total of three major Klebsiella pneumoniae clones (CC14, ST231, and CC147) were identified, accounting for 48.6% of all CRE. All clones were significantly more resistant than sporadic isolates. CC14 strains exhibited a significant association with Emirati patients. CONCLUSIONS: Nearly half of CRE infections in the country are due to a limited number of clones. The data indicate the possibility of interhospital transmission, combined in some hospitals with inadequate stewardship practices. The study also revealed an association of the largest, most resistant clone (CC14) with Emirati patients. The specific reasons for it should be clarified by further investigations.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVES: To assess the current prevalence, distribution, and main clonal types of carbapenem-resistant Enterobacterales (CRE) in the United Arab Emirates. METHODS: A total of 504 CRE collected over a 9-month period in 15 hospitals were studied. Antibiotic susceptibility and the presence of common carbapenemase, 16S methylase, and mobile colistin resistance genes were assessed. Selected strains forming larger clusters by pulsed field gel electrophoresis were subjected to whole genome sequencing to identify their sequence types and core genome MLST. RESULTS: Strains expressing OXA and NDM type carbapenemases and 16S methylases were present in all major hospitals. Considerable interhospital differences were noticed, suggesting the role of specific clones. A total of three major Klebsiella pneumoniae clones (CC14, ST231, and CC147) were identified, accounting for 48.6% of all CRE. All clones were significantly more resistant than sporadic isolates. CC14 strains exhibited a significant association with Emirati patients. CONCLUSIONS: Nearly half of CRE infections in the country are due to a limited number of clones. The data indicate the possibility of interhospital transmission, combined in some hospitals with inadequate stewardship practices. The study also revealed an association of the largest, most resistant clone (CC14) with Emirati patients. The specific reasons for it should be clarified by further investigations.
Sonnevend, Á; Alali, W. Q.; Mahmoud, S. A.; Ghazawi, A.; Bharathan, G.; Melegh, S.; Rizvi, T. A.; Pál, T.
Molecular Characterization of MCR-1 Producing Enterobacterales Isolated in Poultry Farms in the United Arab Emirates Journal Article
In: Antibiotics (Basel), vol. 11, no. 3, 2022, ISSN: 2079-6382 (Print) 2079-6382.
@article{RN31,
title = {Molecular Characterization of MCR-1 Producing Enterobacterales Isolated in Poultry Farms in the United Arab Emirates},
author = {Á Sonnevend and W. Q. Alali and S. A. Mahmoud and A. Ghazawi and G. Bharathan and S. Melegh and T. A. Rizvi and T. Pál},
doi = {10.3390/antibiotics11030305},
issn = {2079-6382 (Print) 2079-6382},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Antibiotics (Basel)},
volume = {11},
number = {3},
abstract = {Data on the prevalence of MCR-producing Enterobacterales of animal origin are scarce from the Arabian Peninsula. We investigated the presence and variety of such strains from fecal specimens of poultry collected in four farms in the United Arab Emirates. Colonies from ten composite samples per farm grown on colistin-supplemented plates were PCR-screened for alleles of the mcr gene. Thirty-nine isolates selected based on species, colony morphology, and plasmid profile were subjected to whole genome sequencing. The panel of their resistance and virulence genes, MLST and cgMLST were established. Transferability and incompatibility types of the MCR-plasmids were determined. mcr-1.1 positive strains were identified in 36 of the 40 samples. Thirty-four multi-drug resistant Escherichia coli of 16 different sequence types, two Escherichia albertii, two Klebsiella pneumoniae and one Salmonella minnesota were identified. Beyond various aminoglycoside, tetracycline, and co-trimoxazole resistance genes, seven of them also carried ESBL genes and one bla(CMY-2). Six IncHI2, 26 IncI2 and 4 IncX4 MCR-plasmids were mobilized, in case of the IncHI2 plasmids co-transferring ampicillin, chloramphenicol and tetracycline resistance. The diversity of mcr-1 positive strains suggest a complex local epidemiology calling for a coordinated surveillance including animals, retail meat and clinical cases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Data on the prevalence of MCR-producing Enterobacterales of animal origin are scarce from the Arabian Peninsula. We investigated the presence and variety of such strains from fecal specimens of poultry collected in four farms in the United Arab Emirates. Colonies from ten composite samples per farm grown on colistin-supplemented plates were PCR-screened for alleles of the mcr gene. Thirty-nine isolates selected based on species, colony morphology, and plasmid profile were subjected to whole genome sequencing. The panel of their resistance and virulence genes, MLST and cgMLST were established. Transferability and incompatibility types of the MCR-plasmids were determined. mcr-1.1 positive strains were identified in 36 of the 40 samples. Thirty-four multi-drug resistant Escherichia coli of 16 different sequence types, two Escherichia albertii, two Klebsiella pneumoniae and one Salmonella minnesota were identified. Beyond various aminoglycoside, tetracycline, and co-trimoxazole resistance genes, seven of them also carried ESBL genes and one bla(CMY-2). Six IncHI2, 26 IncI2 and 4 IncX4 MCR-plasmids were mobilized, in case of the IncHI2 plasmids co-transferring ampicillin, chloramphenicol and tetracycline resistance. The diversity of mcr-1 positive strains suggest a complex local epidemiology calling for a coordinated surveillance including animals, retail meat and clinical cases.
2021
Mouftah, S. F.; Pál, T.; Higgins, P. G.; Ghazawi, A.; Idaghdour, Y.; Alqahtani, M.; Omrani, A. S.; Rizvi, T. A.; Sonnevend, Á
Diversity of carbapenem-resistant Klebsiella pneumoniae ST14 and emergence of a subgroup with KL64 capsular locus in the Arabian Peninsula Journal Article
In: Eur J Clin Microbiol Infect Dis, 2021, ISSN: 0934-9723.
@article{RN92,
title = {Diversity of carbapenem-resistant Klebsiella pneumoniae ST14 and emergence of a subgroup with KL64 capsular locus in the Arabian Peninsula},
author = {S. F. Mouftah and T. Pál and P. G. Higgins and A. Ghazawi and Y. Idaghdour and M. Alqahtani and A. S. Omrani and T. A. Rizvi and Á Sonnevend},
doi = {10.1007/s10096-021-04384-2},
issn = {0934-9723},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Eur J Clin Microbiol Infect Dis},
abstract = {To understand the reasons of successful spread of carbapenem-resistant Klebsiella pneumoniae ST14 (CRKP-ST14) in countries of the Arabian Peninsula, the resistome, capsular locus, carbapenemase carrying plasmid types, and core genome of isolates from the region were compared to global isolates. Thirty-nine CRKP-ST14 strains isolated from 13 hospitals in the United Arab Emirates, Bahrain, and Saudi Arabia were selected for whole genome sequencing on Illumina MiSeq platform based on the variety of carbapenemase genes carried and plasmids bearing these genes. Their resistome, capsular locus, and core genome MLST were compared to 173 CRKP-ST14 genomes available in public databases. The selected 39 CRKP-ST14 produced either NDM-1, OXA-48, OXA-162, OXA-232, KPC-2, or co-produced NDM-1 and an OXA-48-like carbapenemase. cgMLST revealed three clusters: 16 isolates from five UAE cities (C1), 11 isolates from three UAE cities and Bahrain (C2), and 5 isolates from Saudi Arabia (C3), respectively, and seven singletons. Resistance gene profile, carbapenemase genes, and their plasmid types were variable in both C1 and C2 clusters. The majority of CRKP-ST14 had KL2, but members of the C2 cluster and two further singletons possessed KL64 capsular locus. Based on cgMLST comparison of regional and global isolates, CRKP-ST14 with KL64 from four continents formed a distinct cluster, suggesting a recent emergence and spread of this variant. Our findings confirmed clonal transmission coupled with likely horizontal gene transfer in carbapenem-resistant Klebsiella pneumoniae ST14. Dissemination of this genetically flexible, highly resistant clone warrants further monitoring.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
To understand the reasons of successful spread of carbapenem-resistant Klebsiella pneumoniae ST14 (CRKP-ST14) in countries of the Arabian Peninsula, the resistome, capsular locus, carbapenemase carrying plasmid types, and core genome of isolates from the region were compared to global isolates. Thirty-nine CRKP-ST14 strains isolated from 13 hospitals in the United Arab Emirates, Bahrain, and Saudi Arabia were selected for whole genome sequencing on Illumina MiSeq platform based on the variety of carbapenemase genes carried and plasmids bearing these genes. Their resistome, capsular locus, and core genome MLST were compared to 173 CRKP-ST14 genomes available in public databases. The selected 39 CRKP-ST14 produced either NDM-1, OXA-48, OXA-162, OXA-232, KPC-2, or co-produced NDM-1 and an OXA-48-like carbapenemase. cgMLST revealed three clusters: 16 isolates from five UAE cities (C1), 11 isolates from three UAE cities and Bahrain (C2), and 5 isolates from Saudi Arabia (C3), respectively, and seven singletons. Resistance gene profile, carbapenemase genes, and their plasmid types were variable in both C1 and C2 clusters. The majority of CRKP-ST14 had KL2, but members of the C2 cluster and two further singletons possessed KL64 capsular locus. Based on cgMLST comparison of regional and global isolates, CRKP-ST14 with KL64 from four continents formed a distinct cluster, suggesting a recent emergence and spread of this variant. Our findings confirmed clonal transmission coupled with likely horizontal gene transfer in carbapenem-resistant Klebsiella pneumoniae ST14. Dissemination of this genetically flexible, highly resistant clone warrants further monitoring.
2020
Sonnevend, Á; Ghazawi, A.; Darwish, D.; Barathan, G.; Hashmey, R.; Ashraf, T.; Rizvi, T. A.; Pál, T.
In: Int J Infect Dis, vol. 99, pp. 253-259, 2020, ISSN: 1201-9712.
@article{RN81,
title = {In vitro efficacy of ceftazidime-avibactam, aztreonam-avibactam and other rescue antibiotics against carbapenem-resistant Enterobacterales from the Arabian Peninsula},
author = {Á Sonnevend and A. Ghazawi and D. Darwish and G. Barathan and R. Hashmey and T. Ashraf and T. A. Rizvi and T. Pál},
doi = {10.1016/j.ijid.2020.07.050},
issn = {1201-9712},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Int J Infect Dis},
volume = {99},
pages = {253-259},
abstract = {OBJECTIVES: Our aim was to assess the susceptibility of carbapenem-resistant Enterobacterales (CRE) from the Arabian Peninsula to a broad spectrum of antibiotics, including fosfomycin, ceftazidime-avibactam, and aztreonam-avibactam. METHODS: 1192 non-repeat CRE isolated in 2009-2017 from 33 hospitals in five countries of the Arabian Peninsula were tested. The minimum inhibitory concentration of 14 antibiotics was determined. Carbapenemase and 16S methylase genes were detected by PCR. Clonality was assessed by PFGE. RESULTS: The highest rate of susceptibility was detected to aztreonam-avibactam (95.5%) followed by colistin (79.8%), fosfomycin (71.8%) and tigecycline (59.9%). Isolates co-producing two carbapenemases (12.4%) were the least susceptible. Aminoglycoside susceptibility was affected by the frequent production of a 16S methylase. Susceptibility to ceftazidime-avibactam was impacted by the high rate of metallo-beta-lactamase producers (46.3%), while aztreonam-avibactam resistance occurred mostly in clonally unrelated, carbapenemase non-producing Escherichia coli. CONCLUSION: Of the currently available drugs: colistin, tigecycline, and ceftazidime-avibactam co-administered with aztreonam appear to be the most effective to treat CRE infections. However, the presence of non-clonal CRE isolates, in which avibactam does not lower the aztreonam MIC below the clinical breakpoint, is of notable concern. Based on the relatively high rate of fosfomycin susceptibility, it would be desirable to license parenteral fosfomycin in the region.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVES: Our aim was to assess the susceptibility of carbapenem-resistant Enterobacterales (CRE) from the Arabian Peninsula to a broad spectrum of antibiotics, including fosfomycin, ceftazidime-avibactam, and aztreonam-avibactam. METHODS: 1192 non-repeat CRE isolated in 2009-2017 from 33 hospitals in five countries of the Arabian Peninsula were tested. The minimum inhibitory concentration of 14 antibiotics was determined. Carbapenemase and 16S methylase genes were detected by PCR. Clonality was assessed by PFGE. RESULTS: The highest rate of susceptibility was detected to aztreonam-avibactam (95.5%) followed by colistin (79.8%), fosfomycin (71.8%) and tigecycline (59.9%). Isolates co-producing two carbapenemases (12.4%) were the least susceptible. Aminoglycoside susceptibility was affected by the frequent production of a 16S methylase. Susceptibility to ceftazidime-avibactam was impacted by the high rate of metallo-beta-lactamase producers (46.3%), while aztreonam-avibactam resistance occurred mostly in clonally unrelated, carbapenemase non-producing Escherichia coli. CONCLUSION: Of the currently available drugs: colistin, tigecycline, and ceftazidime-avibactam co-administered with aztreonam appear to be the most effective to treat CRE infections. However, the presence of non-clonal CRE isolates, in which avibactam does not lower the aztreonam MIC below the clinical breakpoint, is of notable concern. Based on the relatively high rate of fosfomycin susceptibility, it would be desirable to license parenteral fosfomycin in the region.
2012
Ghazawi, A.; Sonnevend, A.; Bonnin, R. A.; Poirel, L.; Nordmann, P.; Hashmey, R.; Rizvi, T. A.; M, B. Hamadeh; Pál, T.
NDM-2 carbapenemase-producing Acinetobacter baumannii in the United Arab Emirates Journal Article
In: Clin Microbiol Infect, vol. 18, no. 2, pp. E34-6, 2012, ISSN: 1198-743x.
@article{RN58,
title = {NDM-2 carbapenemase-producing Acinetobacter baumannii in the United Arab Emirates},
author = {A. Ghazawi and A. Sonnevend and R. A. Bonnin and L. Poirel and P. Nordmann and R. Hashmey and T. A. Rizvi and B. Hamadeh M and T. Pál},
doi = {10.1111/j.1469-0691.2011.03726.x},
issn = {1198-743x},
year = {2012},
date = {2012-01-01},
urldate = {2012-01-01},
journal = {Clin Microbiol Infect},
volume = {18},
number = {2},
pages = {E34-6},
abstract = {Screening 155 carbapenem non-susceptible Acinetobacter baumannii strains recovered in Abu Dhabi hospitals identified two metallo-ß-lactamase bla(NDM) gene-carrying isolates. They were isolated 4 months apart from the urine of a cancer patient previously treated in Egypt, Lebanon and in the United Arab Emirates. They were clonally related and carried the bla(NDM-2) gene recently identified in A. baumannii in Egypt and Israel. Sequences surrounding the bla(NDM-2) gene showed significant similarities with those associated with bla(NDM-1) in Enterobacteriaceae and A. baumannii. Repeated isolation of bla(NDM-2)-positive A. baumannii in the Middle East raises the possibility of the local emergence and spread of a unique clone.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Screening 155 carbapenem non-susceptible Acinetobacter baumannii strains recovered in Abu Dhabi hospitals identified two metallo-ß-lactamase bla(NDM) gene-carrying isolates. They were isolated 4 months apart from the urine of a cancer patient previously treated in Egypt, Lebanon and in the United Arab Emirates. They were clonally related and carried the bla(NDM-2) gene recently identified in A. baumannii in Egypt and Israel. Sequences surrounding the bla(NDM-2) gene showed significant similarities with those associated with bla(NDM-1) in Enterobacteriaceae and A. baumannii. Repeated isolation of bla(NDM-2)-positive A. baumannii in the Middle East raises the possibility of the local emergence and spread of a unique clone.
2010
Rizvi, T. A.; Kenyon, J. C.; Ali, J.; Aktar, S. J.; Phillip, P. S.; Ghazawi, A.; Mustafa, F.; Lever, A. M. L.
Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag Journal Article
In: J Mol Biol, vol. 403, no. 1, pp. 103-119, 2010, ISSN: 0022-2836 (Print) 0022-2836.
@article{RN49,
title = {Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag},
author = {T. A. Rizvi and J. C. Kenyon and J. Ali and S. J. Aktar and P. S. Phillip and A. Ghazawi and F. Mustafa and A. M. L. Lever},
doi = {10.1016/j.jmb.2010.08.019},
issn = {0022-2836 (Print) 0022-2836},
year = {2010},
date = {2010-01-01},
urldate = {2010-01-01},
journal = {J Mol Biol},
volume = {403},
number = {1},
pages = {103-119},
abstract = {The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.
2009
Rizvi, T. A.; Ali, J.; Phillip, P. S.; Ghazawi, A.; Jayanth, P.; Mustafa, F.
In: Virology, vol. 385, no. 2, pp. 464-72, 2009, ISSN: 0042-6822.
@article{RN94,
title = {Role of a heterologous retroviral transport element in the development of genetic complementation assay for mouse mammary tumor virus (MMTV) replication},
author = {T. A. Rizvi and J. Ali and P. S. Phillip and A. Ghazawi and P. Jayanth and F. Mustafa},
doi = {10.1016/j.virol.2008.12.027},
issn = {0042-6822},
year = {2009},
date = {2009-01-01},
urldate = {2009-01-01},
journal = {Virology},
volume = {385},
number = {2},
pages = {464-72},
abstract = {The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.
Dhaheri, N. S. Al; Phillip, P. S.; Ghazawi, A.; Ali, J.; Beebi, E.; Jaballah, S. A.; Rizvi, T. A.
Cross-packaging of genetically distinct mouse and primate retroviral RNAs Journal Article
In: Retrovirology, vol. 6, pp. 66, 2009, ISSN: 1742-4690.
@article{RN66,
title = {Cross-packaging of genetically distinct mouse and primate retroviral RNAs},
author = {N. S. Al Dhaheri and P. S. Phillip and A. Ghazawi and J. Ali and E. Beebi and S. A. Jaballah and T. A. Rizvi},
doi = {10.1186/1742-4690-6-66},
issn = {1742-4690},
year = {2009},
date = {2009-01-01},
urldate = {2009-01-01},
journal = {Retrovirology},
volume = {6},
pages = {66},
abstract = {BACKGROUND: The mouse mammary tumor virus (MMTV) is unique from other retroviruses in having multiple viral promoters, which can be regulated by hormones in a tissue specific manner. This unique property has lead to increased interest in studying MMTV replication with the hope of developing MMTV based vectors for human gene therapy. However, it has recently been reported that related as well as unrelated retroviruses can cross-package each other's genome raising safety concerns towards the use of candidate retroviral vectors for human gene therapy. Therefore, using a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV) that has intracellular assembly process similar to that of MMTV. RESULTS: Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the heterologous virus particles reciprocally suggesting that pseudotyping between two genetically distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To further confirm that the specificity of cross-packaging was conferred by the packaging sequences (psi), we cloned the packaging sequences of these viruses on expression plasmids that generated non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was primarily due to the recognition of psi by the heterologous virus proteins. CONCLUSION: The results presented in this study strongly argue that MPMV and MMTV are promiscuous in their ability to cross-package each other's genome suggesting potential RNA-protein interactions among divergent retroviral RNAs proposing that these interactions are more complicated than originally thought. Furthermore, these observations raise the possibility that MMTV and MPMV genomes could also co-package providing substrates for exchanging genetic information.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: The mouse mammary tumor virus (MMTV) is unique from other retroviruses in having multiple viral promoters, which can be regulated by hormones in a tissue specific manner. This unique property has lead to increased interest in studying MMTV replication with the hope of developing MMTV based vectors for human gene therapy. However, it has recently been reported that related as well as unrelated retroviruses can cross-package each other's genome raising safety concerns towards the use of candidate retroviral vectors for human gene therapy. Therefore, using a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV) that has intracellular assembly process similar to that of MMTV. RESULTS: Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the heterologous virus particles reciprocally suggesting that pseudotyping between two genetically distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To further confirm that the specificity of cross-packaging was conferred by the packaging sequences (psi), we cloned the packaging sequences of these viruses on expression plasmids that generated non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was primarily due to the recognition of psi by the heterologous virus proteins. CONCLUSION: The results presented in this study strongly argue that MPMV and MMTV are promiscuous in their ability to cross-package each other's genome suggesting potential RNA-protein interactions among divergent retroviral RNAs proposing that these interactions are more complicated than originally thought. Furthermore, these observations raise the possibility that MMTV and MPMV genomes could also co-package providing substrates for exchanging genetic information.
2008
Kenyon, J. C.; Ghazawi, A.; Cheung, W. K.; Phillip, P. S.; Rizvi, T. A.; Lever, A. M.
In: Rna, vol. 14, no. 12, pp. 2597-608, 2008, ISSN: 1355-8382 (Print) 1355-8382.
@article{RN60,
title = {The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop},
author = {J. C. Kenyon and A. Ghazawi and W. K. Cheung and P. S. Phillip and T. A. Rizvi and A. M. Lever},
doi = {10.1261/rna.1284908},
issn = {1355-8382 (Print) 1355-8382},
year = {2008},
date = {2008-01-01},
urldate = {2008-01-01},
journal = {Rna},
volume = {14},
number = {12},
pages = {2597-608},
abstract = {Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.
2006
Ghazawi, A.; Mustafa, F.; Phillip, P. S.; Jayanth, P.; Ali, J.; Rizvi, T. A.
In: Microbes Infect, vol. 8, no. 3, pp. 767-78, 2006, ISSN: 1286-4579 (Print) 1286-4579.
@article{RN118,
title = {Both the 5' and 3' LTRs of FIV contain minor RNA encapsidation determinants compared to the two core packaging determinants within the 5' untranslated region and gag},
author = {A. Ghazawi and F. Mustafa and P. S. Phillip and P. Jayanth and J. Ali and T. A. Rizvi},
doi = {10.1016/j.micinf.2005.09.015},
issn = {1286-4579 (Print) 1286-4579},
year = {2006},
date = {2006-01-01},
urldate = {2006-01-01},
journal = {Microbes Infect},
volume = {8},
number = {3},
pages = {767-78},
abstract = {This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.
2005
Mustafa, F.; Ghazawi, A.; Jayanth, P.; Phillip, P. S.; Ali, J.; Rizvi, T. A.
In: J Virol, vol. 79, no. 21, pp. 13817-21, 2005, ISSN: 0022-538X (Print) 0022-538x.
@article{RN121,
title = {Sequences intervening between the core packaging determinants are dispensable for maintaining the packaging potential and propagation of feline immunodeficiency virus transfer vector RNAs},
author = {F. Mustafa and A. Ghazawi and P. Jayanth and P. S. Phillip and J. Ali and T. A. Rizvi},
doi = {10.1128/jvi.79.21.13817-13821.2005},
issn = {0022-538X (Print) 0022-538x},
year = {2005},
date = {2005-01-01},
urldate = {2005-01-01},
journal = {J Virol},
volume = {79},
number = {21},
pages = {13817-21},
abstract = {The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to approximately 150 bp of the 5' untranslated region and the first approximately 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to approximately 150 bp of the 5' untranslated region and the first approximately 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.
Mustafa, F.; Jayanth, P.; Phillip, P. S.; Ghazawi, A.; Schmidt, R. D.; Lew, K. A.; Rizvi, T. A.
Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines Journal Article
In: Microbes Infect, vol. 7, no. 2, pp. 233-9, 2005, ISSN: 1286-4579 (Print) 1286-4579.
@article{RN139,
title = {Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines},
author = {F. Mustafa and P. Jayanth and P. S. Phillip and A. Ghazawi and R. D. Schmidt and K. A. Lew and T. A. Rizvi},
doi = {10.1016/j.micinf.2004.10.015},
issn = {1286-4579 (Print) 1286-4579},
year = {2005},
date = {2005-01-01},
urldate = {2005-01-01},
journal = {Microbes Infect},
volume = {7},
number = {2},
pages = {233-9},
abstract = {The feline immunodeficiency virus (FIV) long terminal repeat (LTR), compared with some primate lentiviral LTRs, is quite a strong basal promoter. However, it seems to be highly species-specific in function and generally not very efficient in cells of non-feline origin. This study systematically explored the function of the FIV LTR in simian Cos cells compared with its activity in feline and human cells. Our studies, using biologically relevant two- and three-plasmid trans complementation assays followed by semi-quantitative reverse transcriptase PCR, show that the FIV LTR is functional in Cos cells. The results of the Cos experiment are different from previously reported literature and suggest that the strain specificity of the FIV LTR is an important determinant of whether the LTR will be functional in a particular cell type.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The feline immunodeficiency virus (FIV) long terminal repeat (LTR), compared with some primate lentiviral LTRs, is quite a strong basal promoter. However, it seems to be highly species-specific in function and generally not very efficient in cells of non-feline origin. This study systematically explored the function of the FIV LTR in simian Cos cells compared with its activity in feline and human cells. Our studies, using biologically relevant two- and three-plasmid trans complementation assays followed by semi-quantitative reverse transcriptase PCR, show that the FIV LTR is functional in Cos cells. The results of the Cos experiment are different from previously reported literature and suggest that the strain specificity of the FIV LTR is an important determinant of whether the LTR will be functional in a particular cell type.
2004
Mustafa, F.; Phillip, P. S.; Jayanth, P.; Ghazawi, A.; Lew, K. A.; Schmidt, R. D.; Rizvi, T. A.
Close proximity of the MPMV CTE to the polyadenylation sequences is important for efficient function in the subgenomic context Journal Article
In: Virus Res, vol. 105, no. 2, pp. 209-18, 2004, ISSN: 0168-1702 (Print) 0168-1702.
@article{RN143,
title = {Close proximity of the MPMV CTE to the polyadenylation sequences is important for efficient function in the subgenomic context},
author = {F. Mustafa and P. S. Phillip and P. Jayanth and A. Ghazawi and K. A. Lew and R. D. Schmidt and T. A. Rizvi},
doi = {10.1016/j.virusres.2004.06.014},
issn = {0168-1702 (Print) 0168-1702},
year = {2004},
date = {2004-01-01},
urldate = {2004-01-01},
journal = {Virus Res},
volume = {105},
number = {2},
pages = {209-18},
abstract = {The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) is a short cis-acting sequence element critical for virus gene expression. Analogous to the Rev/Rev Responsive Element (RRE) of primate lentiviruses, CTE allows the nucleocytoplasmic transport of unspliced viral mRNAs. In fact, CTE can functionally replace Rev/RRE in the genomic context and has been used successfully in the expression of viral and cellular genes from expression vectors as well. However, unlike RRE, CTE accomplishes this by interacting with cellular factors, making CTE function independent of co-expressed trans factors. Thus, CTE has proven to be a valuable tool in the expression of heterologous genes. Our previous studies have shown that close proximity of CTE to the polyadenylation sequences is important for CTE function in the genomic context. However, it is controversial whether CTE needs to be located spatially close to the polyadenylation sequences in the subgenomic context. Since CTE is being frequently used in expression vectors, we investigated the position dependency of CTE in the heterologous, subgenomic background using both genetic and structural analyses. Our results reveal that similar to the genomic situation, close proximity of CTE to the polyadenylation sequences is important for its function in the heterologous subgenomic context.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) is a short cis-acting sequence element critical for virus gene expression. Analogous to the Rev/Rev Responsive Element (RRE) of primate lentiviruses, CTE allows the nucleocytoplasmic transport of unspliced viral mRNAs. In fact, CTE can functionally replace Rev/RRE in the genomic context and has been used successfully in the expression of viral and cellular genes from expression vectors as well. However, unlike RRE, CTE accomplishes this by interacting with cellular factors, making CTE function independent of co-expressed trans factors. Thus, CTE has proven to be a valuable tool in the expression of heterologous genes. Our previous studies have shown that close proximity of CTE to the polyadenylation sequences is important for CTE function in the genomic context. However, it is controversial whether CTE needs to be located spatially close to the polyadenylation sequences in the subgenomic context. Since CTE is being frequently used in expression vectors, we investigated the position dependency of CTE in the heterologous, subgenomic background using both genetic and structural analyses. Our results reveal that similar to the genomic situation, close proximity of CTE to the polyadenylation sequences is important for its function in the heterologous subgenomic context.