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1.
J Virol ; 97(4): e0140622, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37022163

RESUMO

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.


Assuntos
Cosmídeos , Herpesvirus Galináceo 1 , Mutagênese , Plasmídeos , Animais , Masculino , Galinhas , Cosmídeos/genética , Infecções por Herpesviridae/virologia , Herpesvirus Galináceo 1/genética , Herpesvirus Galináceo 1/patogenicidade , Plasmídeos/genética , Doenças das Aves Domésticas/virologia , Saccharomyces cerevisiae/genética , Linhagem Celular , Genoma Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Genomics ; 112(1): 528-544, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30974149

RESUMO

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.


Assuntos
Mutação , Salmonella enteritidis/genética , Algoritmos , Animais , Fazendas , Genoma Bacteriano , Mutação INDEL , Camundongos , Repetições Minissatélites , Fenótipo , Filogenia , Polimorfismo de Nucleotídeo Único , Aves Domésticas , Salmonella enteritidis/classificação , Salmonella enteritidis/isolamento & purificação , Sequenciamento Completo do Genoma
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