Combined genetic variants of human cytomegalovirus envelope glycoproteins as congenital infection markers

Abstract

Background: Human cytomegalovirus (HCMV) is still considered to be the main viral cause of birth defects and long-term neurological and sensory sequelae following congenital infection. Several Authors sustain a key role of HCMV envelope glycoproteins, such as gB, gN and gO - mainly involved in cell targeting, viral penetration and spread - as putative virulence factors. The genes coding for these glycoproteins possess hypervariable regions, resulting in a number of genetic variants in circulating clinical strains. Considering that the genetic polymorphisms underlying the specific differences between gB, gN and gO genotypes can influence the ability of HCMV to preferentially target specific host cells, it is very likely that they play an important role in defining HCMV infection outcome. In the present study, we analysed HCMV gB, gN and gO gene polymorphisms in viral strains isolated from paediatric patients with congenital or post-natal infection, to investigate whether specific genetic variants may be associated with congenital infection.

Methods: The restriction fragment polymorphisms of genes coding for HCMV gB (UL55), gN (UL73) and gO (UL74) were investigated by analysing viral DNA extracted from 40 urine samples of as many paediatric patients with congenital or post-natal HCMV infection. Randomly selected samples were subjected to DNA sequencing and phylogenetic analysis. Statistical analysis was performed using Fisher's exact test to assess the significance of single and combined glycoprotein genotypes frequency distribution. Statistical significance was considered at a P <0.05.

Results: While gB genomic variants were quite homogeneously represented in both paediatric groups, the gN4 genotype significantly prevailed in congenitally infected children (89.5 %) vs post-natally infected children (47.6 %), with a predominance of the gN4c variant (47.4 %). A similar trend was observed for gO3 (52.6 % vs 19 %). Concerning genotypes association, a statistically significant (P = 0.037) gN4-gO3 combination was found specifically in the congenitally infected group.

Conclusions: The results indicate that the gN4 (mostly the gN4c variant) and gO3 combined genotypes could provide useful markers of congenital infection and represent suitable candidate molecules for prophylactic vaccine preparations.

Fig. 1

Representative RFLP patterns of HCMV gB, gN and gO glycoproteins in the studied population. RFLP analysis was performed on the PCR-amplified gB (a), gN (b) and gO (c) sequences obtained from the patient-derived HCMV strains. Each sample was digested with restriction enzyme combinations as detailed in Tables 1, 2 and 3. In the upper side of panels a (gB) and b (gN), the numbers “1, 2” (panel a), and “1, 2, 3” (panel b) indicate the restriction enzymes used (gB: 1 = RsaI; 2 = HinfI; gN: 1 = SacI; 2 = ScaI; 3 = SalI). A single digestion with HpaII was used to characterise the gO gene products (panel c). The genotypes corresponding to each digest are displayed at the top of the lanes. Lanes MW molecular weight markers

Fig. 2

Phylogenetic analysis of HCMV gB, gN and gO sequences from congenitally and post-natally infected children. Phylogenetic analysis was based on partial UL55 (496 nucleotides) (panel a), UL73 (313 nucleotides) (panel b) and UL74 (275 nucleotides) (panel c). Bootstrap values >60 % are indicated. Scale bars indicate the number of nucleotide substitutions per site. HCMV gB, gN and gO genotypes are indicated on the right side of the dendrograms. Italian strain sequences and reference sequences are indicated with a circle and a triangle, respectively

Fig. 3

Glycoprotein B, N and O genotype distributions in congenitally and post-natally infected children. The overall distributions of gB, gN and gO genotypes are shown in the pie charts of panels a, b and c, respectively; the subdivision of gB, gN and gO variants among congenitally and post-natally infected children is displayed in the adjacent bar charts. The number of subjects infected with the specific genotypes and the related percentages are indicated at the top of each bar

Fig. 4

Combined patterns of HCMV gN and gO genotypes and relationships with clinical pictures at birth. * Premature birth. ** Small size at birth