Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.

Human cytomegalovirus (HCMV) encodes several highly polymorphic envelope glycoproteins; however, the biological relevance of this polymorphism is unclear. Glycoprotein N (gN) is one member of this polymorphic protein family. Four major gN genotypes (gN1-4) have been identified. We have tested the hypothesis that the gN polymorphism represents a mechanism to evade a neutralizing antiviral antibody response. Four recombinant viruses that differed only in the expression of the gN genotype were constructed on the genetic background of HCMV strain AD169. Exchange of gN genotypes had a minor detectable influence on virus replication, gN expression and gN-gM complex formation. Randomly selected human sera were analysed for neutralizing activity against the recombinant viruses. Of these, 70 % showed no difference in neutralizing titre between the viruses, whereas 30 % showed strain-specific neutralization. Differences in 50 % neutralization titre reached .8-fold. Viruses expressing the gN4 genotype were neutralized significantly better than those expressing the other gN genotypes. Strain specificity, or lack thereof, could not be attributed to the presence or absence of anti-gN antibodies, as all sera contained antibodies reacting with gN (as determined by ELISA). Thus, polymorphism of gN could contribute to evasion of an efficient neutralizing-antibody response and facilitate reinfection in previously seropositive individuals. INTRODUCTIONHuman cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals, such as transplant recipients, AIDS patients and newborns . A puzzling aspect of HCMV biology is the fact that a large number of genetically distinct HCMV strains circulate in the population (Chandler & McDougall, 1986;Chou, 1990;Rasmussen et al., 2002Rasmussen et al., , 2003Murphy et al., 2003;Pignatelli et al., 2003). The presence of more than one strain has been observed in immunocompetent persons, indicating the possibility of simultaneous coinfection with multiple different virus strains or reinfection in the presence of a functional immune response (Chandler et al., 1987;Bale et al., 1996;Schoppel et al., 1997;Boppana et al., 2001). In immunocompromised patients, reinfection may represent a clinically important problem, as a de novo adaptive immune response against the reinfecting strain is impaired. Clinical studies in transplant patients have supported the importance of HCMV reinfection in these patients (Grundy et al., 1988; Coaquette et al., 2004;Ishibashi et al., 2007). Moreover, in women who are seropositive for HCMV, reinfection with a different strain can lead to intrauterine transmission and symptomatic congenital infection (Boppana et al., 2001). In the closely related murine cytomegalovirus (MCMV), simultaneous or sequential infection with different virus strains results in mixed infections in immunocompetent mice (Farroway et al., 2005;Gorman et al., 2006). Thus, reinfection with different strains of cytomegalovirus is frequent in both immunocompetent and ...

The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5′ and 3′ ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was packaged successfully, confirming that the motif identified directs the correct packaging of the segment.