Mirosława Studzińska

Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections

Human cytomegalovirus (HCMV) is the most common congenital infection. HCMV strains display genetic variability in different regions. Distribution of HCMV genotypes in the population of congenitally infected newborns from Central Poland and viral load in newborns’ blood is described and discussed. HCMV isolates were analysed by sequencing at three sites on the genome: the UL144 tumour necrosis factor-alpha (TNFα)-like receptor gene, the US28 beta-chemokine receptor gene and the UL55 envelope glycoprotein B (gB) gene. The newborns’ blood was examined for HCMV DNA with a nested (UL144, UL55) or heminested (US28) polymerase chain reaction, and the genotypes were determined by sequence analysis. HCMV DNA was detectable in 25 out of 55 examined newborns born by HCMV-infected mothers (45.5%). The blood viral load in mother–infant pairs was determined. Most of the newborns had identical virus genotype, gB2 (96%), UL144 B1 (88%) and US28 A2 (84%). These genotypes were detected in all newborns with asymptomatic congenital infection. The occurrence of UL144 B1 or US28 A2 genotypes in the babies examined was significant in comparison to other genotypes (p = 0.0002 and p = 0.040 respectively). There was no association between specific gB subtypes in all patients groups (p = 0.463). There was no correlation between HCMV genotypes and the outcome.

Distribution of cytomegalovirus gN variants and associated clinical sequelae in infants

BACKGROUND Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. OBJECTIVES To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. STUDY DESIGN The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. RESULTS Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed (p=0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. CONCLUSION Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns.

TLR9 2848 GA Heterozygotic Status Possibly Predisposes Fetuses and Newborns to Congenital Infection with Human Cytomegalovirus

Background Some single nucleotide polymorphisms (SNP), located in Toll-like receptor (TLR) genes, were reported to be associated with human cytomegalovirus (HCMV) infections. The study was aimed to assess the correlation of SNPs at TLR4 and TLR9 genes with the occurrence of congenital cytomegaly, based on available samples. Methods Reported case-control study included both HCMV infected and non-infected fetuses and newborns. The specimens were classified to the molecular analyses, based on serological features of the recent infection and HCMV DNAemia in body fluids. TLR SNPs were studied, using multiplex nested PCR-RFLP assay, and determined genotypes were confirmed by sequencing. Hardy-Weinberg equilibrium was assessed for the identified genotypes. The linkage disequilibrium was also estimated for TLR4 SNPs. A relationship between the status of TLR genotypes and congenital cytomegaly development was estimated, using a logistic regression model. Results Hardy Weinberg equilibrium was observed for almost all SNPs, both infected and non-infected patients, with exception of TLR4 896 A>G polymorphism in the control group (P≤0.050). TLR4 896 A>G and 1196 C>T SNPs were found in linkage disequilibrium in both study groups (P≤0.050). The CC genotype at TLR4 1196 SNP and the GA variant at TLR9 2848 G>A SNP were significantly associated with HCMV infection (P≤0.050). The risk of congenital cytomegaly was higher in heterozygotes at TLR9 SNP than in the carriers of other genotypic variants at the reported locus (OR 4.81; P≤0.050). The GC haplotype at TLR4 SNPs and GCA variants at TLR4 and TLR9 SNPs were significantly associated with HCMV infection (P≤0.0001). The ACA variants were more frequent among fetuses and neonates with symptomatic, rather than asymptomatic cytomegaly (P≤0.0001). Conclusions TLR4 and TLR9 polymorphisms may contribute to the development of congenital infection with HCMV in fetuses and neonates. The TLR9 2848 GA heterozygotic status possibly predisposes to HCMV infection, increasing the risk of congenital cytomegaly development.

Relationship between toll-like receptor 2 Arg677Trp and Arg753Gln and toll-like receptor 4 Asp299Gly polymorphisms and cytomegalovirus infection

OBJECTIVES The association among specific single-nucleotide polymorphisms (SNPs) in TLR2 (Arg677Trp, Arg753Gln) and TLR4 (Asp299Gln) and human cytomegalovirus (CMV) infection was studied in infants and adults. METHODS The TLR2 and TLR4 polymorphisms were genotyped in 151 patients with CMV infections and in 78 unrelated healthy individuals. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments. The viral load was measured by quantitative real-time PCR. RESULTS Almost all of the patients with CMV infections were wild-type homozygotes without TLR2 and TLR4 polymorphisms. No significant differences in TLR2 and TLR4 polymorphisms were observed between infants with or without CMV infection. Compared with adults with CMV infections, heterozygosity for the TLR2 Arg677Trp and TLR4 Asp299Gly SNPs was detected more frequently in healthy individuals (p<0.05). Logistic regression analysis showed that the wild-type TLR2 genotype was associated with an increased risk of CMV infection and that heterozygosity for TLR2 and TLR4 SNPs diminished the risk of CMV infection in adult patients. An association between CMV load and the TLR4 SNP was found. CONCLUSION Our results suggest that the wild-type TLR2 genotype may be a risk factor for CMV replication in adult patients.

TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants

Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni’s correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease.

Cytomegalovirus alpha-chemokine genotypes are associated with clinical manifestations in children with congenital or postnatal infections.

Human cytomegalovirus (HCMV) is the leading cause of congenital infections. The aim of our study was to determine the prevalence of genotypes based on the highly polymorphic UL146 and UL147 HCMV genes and the relationship between the genotype and symptoms or viral load. We analyzed samples from 121 infants with symptomatic HCMV infection, including 32 congenitally infected newborns. The G7 and G5 genotypes were predominant in postnatal infection, whereas the G1 genotype was prevalent in congenital infection. Central nervous system (CNS) damage and hepatomegaly were detected more frequently among children infected with the G1 genotype than in those infected by other genotypes. An association between the viral genotype and viruria level was found. There was a strong correlation between HCMV genotypes determined through the UL146 and UL147 sequences (ĸ=0.794). In conclusion, we found that certain vCXCL genotypes are associated with clinical sequelae following HCMV infection.

Mannan-binding lectin-2 (MBL2) gene polymorphisms in prenatal and perinatal cytomegalovirus infections

Cytomegalovirus (CMV) is the leading cause of congenital infections among neonates. About 10% of newborns with such an infection have clinical symptoms at birth and about 1% of infected fetuses die due to developmental malformations. Mannan-binding lectin (MBL) is considered to be an important factor in innate immunity. Its deficiency is believed to predispose to various (including viral) infections. The aim of this study was to investigate the possible role of MBL2 gene polymorphisms in prenatal and perinatal CMV infections. The frequencies of MBL2 gene exon 1 mutations as well as MBL deficiency-associated variants (LXPA/O+O/O) among newborns with confirmed cytomegalovirus infection were not significantly lower than among non-infected individuals. The distribution of MBL2 haplotypes was similar between the groups studied. These data suggest MBL does not have a major influence on susceptibility to prenatal or perinatal CMV infections.

Cytomegalovirus glycoprotein H genotype distribution and the relationship with hearing loss in children

Cytomegalovirus (CMV) is a leading cause of congenital infection and a leading infectious cause of hearing loss in children. The ORF UL75 gene encodes envelope glycoprotein H (gH), which is essential for CMV entry into host cells and the target of the immune response in humans. However, the distribution of gH variants and the relationship between the viral genotype, viral load, and sequelae in children infected with CMV is debated. The UL75 genetic variation of CMV isolates from 42 newborns infected congenitally with CMV and 93 infants with postnatal or unproven congenital CMV infection was analyzed. Genotyping was performed by analysis of PCR‐amplified fragments, and the viral load was measured by quantitative real‐time PCR. There were no differences in the distribution of gH genotypes in the children infected congenitally and postnatally. Mixed‐genotype infections with both gH1 and gH2 variants were detected in approximately 25% of the examined patients. No relationship between UL75 gene polymorphisms and the symptoms at birth was observed. The results suggest that the infection with gH2 genotype diminishes the risk of hearing loss in children (P = 0.010). In addition, sensorineural hearing loss was associated with CMV gH1 genotype infection in infants (P = 0.032) and a high viral load in urine (P = 0.005). In conclusion, it was found that the gH genotype does not predict clinical sequelae in newborn infants following congenital CMV infection. However, these results suggest that the gH genotype might be associated with hearing loss in children. J. Med. Virol. 86:1421–1427, 2014.

Toll-like receptors genes polymorphisms and the occurrence of HCMV infection among pregnant women

BackgroundHuman cytomegalovirus (HCMV) is the most common cause of intrauterine infections worldwide. The toll-like receptors (TLRs) have been reported as important factors in immune response against HCMV. Particularly, TLR2, TLR4 and TLR9 have been shown to be involved in antiviral immunity. Evaluation of the role of single nucleotide polymorphisms (SNPs), located within TLR2, TLR4 and TLR9 genes, in the development of human cytomegalovirus (HCMV) infection in pregnant women and their fetuses and neonates, was performed.MethodsThe study was performed for 131 pregnant women, including 66 patients infected with HCMV during pregnancy, and 65 age-matched control pregnant individuals. The patients were selected to the study, based on serological status of anti-HCMV IgG and IgM antibodies and on the presence of viral DNA in their body fluids. Genotypes in TLR2 2258 A > G, TLR4 896 G > A and 1196 C > T and TLR9 2848 G > A SNPs were determined by self-designed nested PCR-RFLP assays. Randomly selected PCR products, representative for distinct genotypes in TLR SNPs, were confirmed by sequencing. A relationship between the genotypes, alleles, haplotypes and multiple variants in the studied polymorphisms, and the occurrence of HCMV infection in pregnant women and their offsprings, was determined, using a logistic regression model.ResultsGenotypes in all the analyzed polymorphisms preserved the Hardy-Weinberg equilibrium in pregnant women, both infected and uninfected with HCMV (P > 0.050). GG homozygotic and GA heterozygotic status in TLR9 2848 G > A SNP decreased significantly the occurrence of HCMV infection (OR 0.44 95% CI 0.21–0.94 in the dominant model, P ≤ 0.050). The G allele in TLR9 SNP was significantly more frequent among the uninfected pregnant women than among the infected ones (χ2 = 4.14, P ≤ 0.050). Considering other polymorphisms, similar frequencies of distinct genotypes, haplotypes and multiple-SNP variants were observed between the studied groups of patients.ConclusionsTLR9 2848 G > A SNP may be associated with HCMV infection in pregnant women.

TLR2 2258 G>A single nucleotide polymorphism and the risk of congenital infection with human cytomegalovirus

BackgroundHuman cytomegalovirus (HCMV) is responsible for the most common intrauterine infections, which may be acquired congenitally from infected pregnant woman to fetus. The research was aimed to estimate the role of three single nucleotide polymorphisms (SNPs) located in TLR2 gene, and the common contribution of TLR2, and previously studied TLR4 and TLR9 SNPs, to the occurrence of congenital HCMV infection in fetuses and newborns.MethodsThe study was performed in 20 Polish fetuses and newborns, congenitally infected with HCMV, and in 31 uninfected controls, as well as with participation of pregnant women, the mothers of 16 infected and 14 uninfected offsprings. Genotypes in TLR2 SNPs were determined, using self-designed nested PCR-RFLP assays, and confirmed by sequencing. The genotypes were tested for Hardy-Weinberg (H-W) equilibrium, and for their relationship with the development of congenital cytomegaly, using a logistic regression model. The common influence of TLR2, TLR4 and TLR9 SNPs on the occurrence of congenital disease was estimated by multiple-SNP analysis.ResultsDistribution of the genotypes and alleles in TLR2 1350 T>C and 2029 C>T SNPs was similar between the studied groups of fetuses and neonates. In case of 2258 G>A polymorphism, the GA heterozygotic status was significantly more frequent in the infected cases than among the uninfected individuals (25.0% vs. 3.2%, respectively), and increased the risk of HCMV infection (OR 10.00, 95% CI 1.07–93.44; P ≤ 0.050). Similarly, the A allele within 2258 G>A polymorphism was significantly more frequent among the infected offsprings than in the uninfected ones (12.5% vs. 1.6%; P ≤ 0.050). Complex AA variants for both TLR2 2258 and TLR9 2848 G>A polymorphisms, were estimated to be at increased risk of congenital HCMV infection (OR 11.58, 95% CI 1.19–112.59; P ≤ 0.050). Additionally, significant relationships were observed between the occurrence of complex AA or GA variants for both TLR2 and TLR9 SNPs and the increased viral loads, determined in fetal amniotic fluids and in maternal blood or urine specimens (P ≤ 0.050).ConclusionsAmong various TLR2, TLR4 and TLR9 polymorphisms, TLR2 2258 G>A SNP seems to be an important factor associated with increased risk of congenital HCMV infection in Polish fetuses and neonates.