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.

Human cytomegalovirus (CMV) is the leading cause of intrauterine viral infection. The association of genetic polymorphisms in some particular genes with the incidence and severity of congenital infection has been controversial. To address this issue, we analyzed the genotypes of the glycoprotein B (gB), UL144 and UL149 genes of CMV clinical strains obtained from 33 congenitally and 31 postnatally infected Japanese children. Our results demonstrated that (1) CMV strains with any combination of genotypes could be vertically transmitted from mother to fetus, potentially causing neurological abnormalities, (2) the gB3 genotype was more prevalent in the congenital cases than in postnatally infected children (P < 0.05), particularly in congenital cases with sensorineural hearing loss (P = 0.009), (3) there was no relationship between gB genotype and viral load in the urine and dried umbilical cord specimens in the congenital cases, and (4) the UL144 and UL149 genotype distributions had no bias for congenial infection. In future studies, it would be interesting to see whether the gB genotypes serve as a prognostic indicator of CMV-associated diseases.

Investigation of sequence polymorphisms in the glycoprotein N (gN; gp4273), gO (gp4274) and gH (gp4275) genes of human cytomegalovirus (HCMV) strains collected from 63 Japanese children revealed that their gO genotype distribution differed slightly from that of Caucasian populations and that there was a significant linkage between the gN and gO genotypes. Linkage of these genotypes in strains obtained from Caucasian populations has been reported, so our similar findings in Japanese infants are consistent with this, and suggest generality of this linkage. Sequence analysis suggests that recombination between two strains of different linkage groups occurred approximately 200 bp upstream of the 39-end of the gO gene. Further studies are required to elucidate differences in biological characteristics among the linkage groups and the selective constraints that maintain the linkage.Human cytomegalovirus (HCMV) infects most people during childhood without clinical symptoms; it is the major viral cause of birth defects and developmental abnormalities. It is also associated with significant morbidity and mortality in immunocompromised individuals. The complete genome of wild-type HCMV strains, such as Merlin, is 236 kb in length, and is predicted to encode 165 genes (Dolan et al., 2004). Genetic characterization of clinical isolates has mainly depended on sequence polymorphisms in the genes that encode viral envelope glycoproteins and cellular homologues (Rasmussen, 1999;Pignatelli et al., 2004). Glycoprotein B (gB), gM, gN, gH, gL and gO are involved in virus entry and egress and are the target molecules recognized by neutralizing antibodies. While extensive sequence variation is found in the gB and gH genes (5-10 %), a greater level is found in the gN and gO genes (40-50 %); the gL and gM genes are highly conserved among clinical strains. To date, the association of a particular genotype with a particular clinical outcome has been controversial (Bale et al., 2000;Barbi et al., 2001;Trincado et al., 2000).We have recently investigated gB, UL144 and UL149 gene polymorphisms and found that the gB3 genotype was more prevalent in congenitally infected individuals with neurological abnormalities (Yan et al., 2008). Recently, a genetic linkage between the gN and gO genes was reported (Mattick et al., 2004). To learn how common this linkage is and whether the linkage group has any correlation with the clinical outcome of congenital infection, we analysed gN, gO and gH gene polymorphisms.HCMV strains were collected from 45 urine and 24 dried umbilical cord specimens obtained from 63 Japanese children, consisting of 32 congenitally and 31 post-natally infected children. Although samples of both materials were collected from six infants, specimens from each infant were handled as a single entity, as specimens from the same infant yielded the same sequence. Eleven of the congenital cases were identified previously by Ogawa et al. (2007) the rest were identified by clinical manifestations. All congenital infections were confirmed b...

Since congenital cytomegalovirus (CMV) infection causes late-onset sequelae, the identification of CMVinfected newborns is important. For this purpose, we established a simple real-time PCR assay using a filter disk. Combined with the collection of urine using filter papers placed in the diaper, this assay can make CMV screening more feasible and cost-effective.Congenital cytomegalovirus (CMV) infection occurs in 0.2 to 1% of all births. In addition to infants with symptomatic cases, a proportion of asymptomatically infected newborns face a significant risk of late-onset sequelae (8,9,12,16,17,22,26). Since the early identification of congenitally infected newborns may lead to early intervention and antiviral treatment options (14), a simple and inexpensive assay for CMV detection is required to implement screening programs for congenital CMV infection (19).In spite of the progress in PCR-based assays, the need for DNA purification from body fluid specimens as well as the labor, costs, and other problems associated with specimen collection, transportation, and storage have limited the convenience of these assays. Although a small volume of urine can be used directly for PCR, inhibitors in urine reduce PCR efficiency (6,7,13,23). Robotic systems that may simplify the DNA purification process (20) are not affordable for every facility. The use of filter papers can resolve the problems associated with collection and storage, as exemplified by the use of dried blood spots (10). Washing filter papers has reduced the amount of inhibitors, which in turn has allowed for conventional PCR on the filter disk (25). Alternatively, DNA samples have been extracted and/or purified from specimens on filters and used for PCR (3,5,18,27). Although dried blood spots can be used for CMV screening (2), the assay may not detect some cases of congenital infection, as blood specimens contain smaller amounts of CMV than urine specimens (4, 11; N. Inoue and S. Koyano, unpublished results).In this study, we developed a real-time PCR assay using urine specimens on filter disks as a template for the reaction and demonstrated the assay's technological potential for screening for congenital CMV infection.First, we selected a filter paper on which PCRs could proceed efficiently (Fig. 1). Commercially available filter papers were spiked with dilutions of purified CMV, and filter disks obtained from the filters were added directly to PCR mixtures. Since Isocode filters allowed more efficient amplification than the others, these filters were used for the following experiments. Next, we found that only instruments equipped with a photomultiplier-tube scanning system (e.g., Stratagene MX3500P) could be used for real-time PCR assays with filter disks in the reaction mixture, due to the fact that instruments using a charge-coupled device camera (e.g., ABI7700) were adversely affected by nonspecific signals from the disks. The optimized real-time PCR conditions were as follows. Fifty microliters of reaction mixture contained 1ϫ Brilliant quantitative P...