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The UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b′ region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b′ region predicts acute endothelial tropism and long-term shedding in vivo in the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b′ region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropism in vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b′ region, rapidly emerged during in vivo replication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuated in vivo and suggest that additional UL/b′ genes, besides the UL128 complex, are required for optimal in vivo CMV replication and dissemination.
IMPORTANCE An effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate the in vivo function of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance of in vivo studies in evaluating human CMV gene function and suggest that additional UL/b′ genes are required for optimal CMV dissemination and transmission.
Limited Dissemination and Shedding of the UL128 Complex-Intact, UL/b′-Defective Rhesus Cytomegalovirus Strain 180.92
Basel T. Assaf, Keith G. Mansfield, Lisa Strelow, Susan V. Westmoreland, Peter A. Barry, Amitinder Kaur
Identification and confirmation of known as well as unknown (bio)chemical entities in ambient mass spectrometry (MS) and MS imaging (MSI) mostly involve accurate mass determination, often in combination with MS/MS or MSn work flows. To further improve structural assignment, additional molecular information is required. Here we present an ambient hydrogen/deuterium exchange (HDX) laser ablation electrospray ionization (LAESI) MS method in which, apart from the accurate mass and MS/MS data, the number of exchangeable protons in (un)known molecules is obtained. While eventually presenting ambient HDX-LAESI-MSI, samples were not preincubated with deuterated solvents, but instead HDX occurred following fusion of ablated sample material with microdroplets generated by ESI of deuterated solvents. Therefore, the degree of HDX was first studied following ablation of nondeuterated sample solutions of melamine and monosaccharides. From these experiments, it was concluded that the set-up used could provide meaningful HDX data in support of molecular structure elucidation by significantly reducing the number of structure options from a measured elemental composition. This reduction was demonstrated with an unknown accurate m/z value obtained in the analysis of an orange slice, reducing the possible number of molecular structures having the same elemental composition by 87% due to the number of H/D exchanges observed. Next, deuterated and nondeuterated MS/MS experiments showed the number of exchangeable protons in the substructures from deuterated neutral losses in the product ion spectra, confirming the compound to be arginine. Finally, the potential of ambient HDX-LAESI-MSI was demonstrated by the imaging of (secondary) plant metabolites in a Phalaenopsis petal.
laser ablation electrospray ionization, hydrogen deuterium exchange, ambient mass spectrometry, structure elucidation, mass spectrometry imaging
Laser Ablation Electrospray Ionization Hydrogen/Deuterium Exchange Ambient Mass Spectrometry Imaging
Fred A. M. G. van Geenen, Frank W. Claassen, Maurice C. R. Franssen, Han Zuilhof, Michel W. F. Nielen
2020 Feb 5
The Hawaiian honeycreepers are an avian adaptive radiation containing many endangered and extinct species. They display a dramatic range of phenotypic variation and are a model system for studies of evolution, conservation, disease dynamics and population genetics. Development of a genome-scale resources for this group would augment the quality of research focusing on Hawaiian honeycreepers and facilitate comparative avian genomic research.
We assembled the genome sequence of a Hawaii amakihi (Hemignathus virens),and identified ~3.9 million single nucleotide polymorphisms (SNPs) in the genome. Using the amakihi genome as a reference, we also identified ~156,000 SNPs in RAD tag (restriction site associated DNA) sequencing of five honeycreeper species (palila [Loxioides bailleui], Nihoa finch [Telespiza ultima], iiwi [Vestiaria coccinea], apapane [Himatione sanguinea], and amakihi). SNPs are distributed throughout the amakihi genome, and the individual sequenced shows several large regions of low heterozygosity on chromosomes 1, 5, 6, 8 and 11. SNPs from RAD tag sequencing were also found throughout the genome but were found to be more densely located on microchromosomes, apparently a result of differential distribution of the particular site recognized by restriction enzyme BseXI.
The amakihi genome sequence will be useful for comparative avian genomics research and provides a significant resource for studies in such areas as disease ecology, evolution, and conservation genetics. The genome sequences will enable mapping of transcriptome data for honeycreepers and comparison of gene sequences between avian taxa. Researchers will be able to use the large number of SNP markers to genotype honeycreepers in regions of interest or across the whole genome. There are enough markers to enable use of methods such as genome-wide association studies (GWAS) that will allow researchers to make connections between phenotypic diversity of honeycreepers and specific genetic variants. Genome-wide markers will also help resolve phylogenetic and population genetic questions in honeycreepers.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1098) contains supplementary material, which is available to authorized users.
Genome, Hawaiian honeycreepers, SNP, RAD tags, Drepanidines, Hemignathus virens
enomic resources for the endangered Hawaiian honeycreepers
Taylor Callicrate, Rebecca Dikow, James W Thomas, James C Mullikin, Erich D Jarvis, Robert C Fleischer, NISC Comparative Sequencing Program