BackgroundAs congenital cytomegalovirus (CMV) infection causes significant clinical consequences not only at birth but also later as neurological sequelae, it is critical to establish a strategy for screening congenitally infected newborns. Previous studies have identified an insufficient sensitivity in screening methods based on the use of dried blood spots (DBSs).ObjectivesTo evaluate the feasibility of the authors' recently developed method for large-scale screening for congenital CMV infection and to identify risk factors for congenital infection.MethodsMore than 21 000 newborns were enrolled at 25 sites in six geographically separate areas of Japan. Urine was collected onto filter cards placed in the diapers, which were then analysed by quantitative PCR using the filter disc directly as a template. Clinical and physical findings of the newborns were extracted from their medical records. CMV strains from the cases and their siblings were genetically compared. Viral loads in DBSs obtained from some of the cases were compared with those in the urine filters.ResultsCongenital CMV infection was identified in 0.31% (95% CI 0.24% to 0.39%) of the newborns, and 30% of the cases (20/66) had typical clinical manifestations and/or showed abnormalities in brain images at birth. Although the positive predictive value of our screening was 94%, the lack of any comparison with a gold standard assay prevented calculation of the negative predictive value. Almost two-thirds of the cases had siblings, a significantly higher frequency than for uninfected newborns. Most of the cases (21/25) excreted CMV strains identical to those of their siblings. CMV DNA was undetectable in three out of 12 retrievable DBS specimens.ConclusionsImplementation of an effective large-scale screening programme for congenital CMV infection is feasible. Siblings are the major risk factor for congenital CMV infection, which emphasises the need for education of mothers-to-be as well as vaccine development.

Glycoproteins M (gM) and N (gN) are well conserved across the herpesvirus family and their involvement in virus penetration and egress is well described, especially for alphaherpesviruses. Because there was no previous study on the homologues of human herpesvirus 8 glycoproteins M (gM8) and N (gN8), we analysed their biochemical and functional characteristics. We found that: (i) gM8 aggregated following heat treatment; (ii) gM8 was a virion component; (iii) gM8 and gN8 were N-glycosylated; (iv) gM8 formed a specific complex with gN8; and (v) gN8 was required for functional processing of gM8. Co-expression of gM8 and gN8 inhibited cell fusion induced either by a combination of herpes simplex virus type 1 glycoproteins or by Molony murine leukaemia virus envelope protein. These results indicate that, in addition to the similar biochemical properties, the fusion inhibition reported previously only for alphaherpesviruses is a function conserved in the gammaherpesvirus subfamily.Herpesviruses encode several glycoproteins; some are unique to a particular subfamily or virus, while others, including glycoprotein B (gB), gH, gL, gM and gN, are conserved throughout the herpesvirus family. These glycoproteins are likely to play essential and similar roles during the infectious cycle. However, their functional similarities cannot be assumed based only on the conservation of sequences and biochemical characteristics. An instructive example in this regard is gB, which was not detectable in the virions of some gammaherpesviruses (Gong & Kieff, 1990; Stewart et al., 1994). Although gMs and gNs of most herpesviruses share several biochemical characteristics in addition to their sequence homologies (Baines & Roizman, 1993;Dijkstra et al., 1996; Jöns et al., 1998;Lake et al., 1998;Mach et al., 2000;MacLean et al., 1993;Osterrieder et al., 1996;Pilling et al., 1994; Wu et al., 1998), differences have been noted in their fundamental properties, specifically, the degree of dispensability for growth in cell culture and effects on penetration and/or egress (Adams et al., 1998;Baines & Roizman, 1991;Dijkstra et al., 1996;Hobom et al., 2000; Jöns et al., 1998; König et al., 2002; Lake & HuttFletcher, 2000;MacLean et al., 1991;Osterrieder et al., 1996; Tischer et al., 2002). Because the properties of these glycoproteins have mainly been studied in alphaherpesviruses, additional studies are required to clarify whether any function found in alphaherpesviruses is applicable to other subfamilies. Neither the gM nor the gN homologue of human herpesvirus 8 (HHV-8) has yet been studied in detail. Therefore, in this study, we analysed their biochemical and functional properties.Expression of HHV-8 gM (gM8) (ORF39) was analysed by immunoblotting of cell lysates prepared from BCBL-1 cells, a primary effusion lymphoma cell line containing latent HHV-8 (Renne et al., 1996). The lytic infection was induced by treatment of the cells with 20 ng 12-Otetradecanoylphorbol 13-acetate (TPA) ml 21. Anti-gM8 serum was prepared by immunization of rabbits with...

Congenital CMV infection is an important cause of severe SNHL, and it has an incidence comparable to that of GJB2-associated SNHL.

To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.Attachment and entry represent the first essential steps of viral replication. Enveloped viruses have evolved two main pathways to mediate their entry into the cells after attachment to cell surface moieties (reviewed in reference 25). The first one, low-pH-dependent pathway, involves endocytosis of viral particles followed by viral-cell membrane fusion in endosomes or lysosomes. This fusion is triggered by an acidic-pH-dependent conformational change of viral glycoprotein(s) and allows release of capsid into the cytoplasm. Entry of vesicular stomatitis virus (VSV), the prototype rhabdovirus, exemplifies this pathway. In contrast, in the second one, pH-independent pathway, viral-cell membrane fusion takes place on the plasma membrane at neutral pH. Most retroviruses and paramyxoviruses use this pathway. The pH-independent entry was also demonstrated for herpesviruses, herpes simplex virus (55), and cytomegalovirus (CMV) (16). However, Epstein-Barr virus (EBV) uses membrane fusion both in endosomes and on the plasma membrane differentially (40).Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is a member of the gammaherpesvirus subfamily and is etiologically associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (reviewed in references 1 and 11). It interacts with target cells by binding of glycoprotein B (gB) and K8.1 with glycosaminoglycans, such as heparan sulfate, on the cell surface (5, 10, 54). Recent studies found that gB also bound to ␣3␤1 integrin through its RGD motif and induced ERK signaling pathway, implicating that ␣3␤1 integrin functions as a cellular receptor for HHV-8 entry (6, 42). One of the major constraints to study the entry mechanisms of HHV-8 is a lack of fully permissive cell lines to conduct traditional virological assays b...

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.

TLR-2 polymorphisms may have some association with congenital CMV infection, although the mechanism underlying this effect remains to be clarified.

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...

Although the use of dried blood spots has been proposed for screening of newborns with congenital cytomegalovirus infection, viral loads in blood were significantly smaller than those in urine (P < 0.001), and DNA recovery from dried blood spots using the thermal shock procedure was inefficient. In contrast, our urine-based screening program identified asymptomatic cases with low viral loads in blood.