A sensitive enzyme-immunochemical staining method was developed for detection of N-glycolylneuraminic acid (NeuGc)-containing glycosphingolipids (GSLs) on silica gel thin-layer chromatography. The procedure consists of immune reaction among NeuGc-containing GSLs, affinity-purified chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG, and the peroxidase reaction using 4-chloro-1-naphthol as a chromogenic substrate. Quantitative determination was achieved by direct densitometric scanning of the enzyme-immunostained spots on the chromatogram. As little as 0.5 pmol of NeuGc-LacCer, NeuGc-nLcOse4Cer, and NeuGc-nLcOse6Cer (0.64-1.0 ng) could be detected with a good signal-to-noise ratio. A semi-linear detector response was observed up to 50 pmol of each GSL. This procedure can be applied easily to other glycolipid antigen systems.
Guinea pig cytomegalovirus (GPCMV) provides a useful model for studies of congenital CMV infection. During characterization of the GPCMV genome sequence, we identified two types of strains in a virus stock purchased from ATCC. One of them, GPCMV/del, lacks a 1.6 kb locus that positionally corresponds to murine CMV (MCMV) M129-M133. Growth of GPCMV/del in cell culture was marginally better than that of the other strain, GPCMV/full, which harbors the 1.6 kb locus. However, in animals infected intraperitoneally with virus stocks containing both strains, GPCMV/full disseminated more efficiently than GPCMV/del, including 200-fold greater viral load in salivary glands. Viral DNA, transcripts of the immediate-early 2 gene homolog, and viral antigens were more abundant in animals infected with GPCMV/full than in those infected with GPCMV/del. Although the observed phenomena have some similarity with the growth properties of MCMV strains defective in mck-1/mck-2(M129/131) and those defective in sgg(M132), no M129-M132 homologs were found in the 1.6 kb locus. Since one of the ORFs in the locus has a weak sequence similarity with HCMV UL130, which relates to cell tropism, further studies will be required to learn the mechanism for efficient GPCMV growth in animal.
We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.
Congenital infection by human cytomegalovirus (HCMV) is a major cause of birth defects and developmental abnormalities. Since guinea pig cytomegalovirus (GPCMV) crosses the placenta and causes infection in utero, GPCMV models are useful for studies of the mechanisms of transplacental transmission. During our characterization of a genomic locus required for GPCMV dissemination in animals, we found that the nucleotide sequence in and around the nearby immediate-early genes in our lineage of GPCMV strain 22122 [designated GPCMV (ATCC-P5)] showed clear differences from that reported previously for the same strain [designated GPCMV (UMN)] passaged extensively in vitro. Since in vitro passaging of HCMV is known to result in genetic alterations, especially in the UL128-UL131A locus, and loss of growth ability in particular cell types, in this study we determined the complete genome sequence of GPCMV (ATCC-P5), which grows efficiently in animals. A total of 359 differences were identified between the genome sequences of GPCMV (UMN) and GPCMV (ATCC-P5), and these resulted in structural differences in 29 protein-encoding regions. In addition, some genes predicted from our analysis but not from GPCMV (UMN) are well conserved among cytomegaloviruses. An additional 18 passages of GPCMV (ATCC-P5) in vitro generated no further marked alterations in these genes or in the locus corresponding to the HCMV UL128-UL131A. Our analyses indicate that the published sequence of GPCMV (UMN) contains a substantial number of sequencing errors and, possibly, some mutations resulting from a long history of passaging in vitro. Our re-evaluation of the genetic content of GPCMV will provide a solid foundation for future studies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.