Cytomegalovirus Genotype Distribution among Congenital and Perinatal Infected Patients with CMV-Associated Thrombocytopenia.

Objective: We determined the prevalence and relationship of glycoprotein B (gB), glycoprotein N (gN), and glycoprotein H (gH) genotypes of cytomegalovirus (CMV) in CMV-associated thrombocytopenia (CAP). Methods: CMV gB, gN, and gH strains were determined by nested PCR and restriction length polymorphism from 24 CAP and 20 asymptomatic CMV infected infants. Results: The order of prevalence was gB1 (70.8%,17/24), gN4 (45.8%,11/24) and gH2 (54.2%,13/24). There was a greater prevalence of gB1(75.0%,15/20), gN4(50.0%,10/20) and gN2 (35.0%,7/20) in moderate to severe infection (p = 0.014 and p = 0.003). By logistic regression, gH2 (p = 0.031) had an elevated risk of thrombocytopenia. Reduced risks of thrombocytopenia were associated with gB2 (p = 0.020), gN1 (p = 0.018) and gN3 (p = 0.008). The most virulent were gB1 (p = 0.033) and gN2 (p = 0.038). Conclusions: There may be a potential association between the gH2 genotype of CMV and infantile thrombocytopenia.

Keywords: CMV- associated thrombocytopenia; glycoprotein B; glycoprotein N; glycoprotein H

Cytomegalovirus (CMV) is a member of the herpesvirus family that causes a wide spectrum of diseases in neonates and infants. It is unique in its ability to cause latent infection with secondary reactivation. In China, the seroprevalence of CMV is approximately 94.7–97.8% in fertile women [[1]]. Systematic review has shown a high prevalence of congenital infection and perinatal infection, partly because of the higher maternal CMV seroprevalence [[2], [4]]. The congenital infection is usually transmitted to the fetus in utero, whereas perinatal infection is transmitted through the placenta or through external sources such as the birth canal, saliva, or breast milk.

It is well-known that CMV can cause hematological disorders such as hemolytic anemia, leukopenia, and thrombocytopenia in infants. Thrombocytopenia associated with active CMV infection is considered CAP, which can be distinguished from primary immune thrombocytopenic purpura.

Several envelope glycoproteins such as UL55 gene encoding glycoprotein B (gB), UL73 gene encoding glycoprotein N (gN), and UL75 gene encoding glycoprotein H (gH) have been evaluated in clinical isolates due to their significant role in tissue tropism and virulence. Their genetic polymorphisms are used to classify CMV strains and may influence the infectivity or pathogenicity of CMV [[5]]. It has been hypothesized that genetic variation among CMV strains may underlie strain-specific clinical manifestations [[6], [8]]. With this in mind, a study of the gB, gN and gH genotypes of CMV strains from CAP infants was undertaken. The distribution of CMV genotypes in previous studies in China and infants with asymptomatic CMV infection was determined to serve as a baseline for direct comparison, as well as for epidemiological interest.

Infants with thrombocytopenia and positive identification of CMV infection treated at two hospitals from January 2015 to December 2019 were included. Their clinical features, laboratory data and treatment course were recorded. Patients with primary immunodeficiency, acquired immune deficiency syndrome, leukemia, or inherited bone marrow failure syndrome were excluded from the ultimate analysis. Additionally, patients suffering from Epstein-Barr virus or other virus infections were also excluded. A group of 20 asymptomatic CMV infected infants from the same period of time was also included in the study.

Congenital infections group includes infants presenting CMV associated symptoms or signs within 14 days of birth. Perinatal infections group includes infants presenting CMV associated symptoms or signs 3–12 weeks after birth [[9]]. Moderately to severely symptomatic CMV disease is defined as multiple manifestations attributable to CMV infection including thrombocytopenia, petechiae, hepatomegaly, splenomegaly, intrauterine growth restriction, hepatitis (raised transaminases or bilirubin), respiratory symptoms, moderate or severe anemia, or central nervous system involvement such as microcephaly orradiographic abnormalities consistent with CMV central nervous system disease. Mildly symptomatic CMV disease is defined as occur with one or two isolated manifestations of CMV infection that are mild and transient (eg, mild hepatomegaly or a single measurement of low platelet count or raised levels of alanine aminotransferase) [[10]].

Patients were tested for CMV infection using serological CMV tests (IgM and IgG), viral culture, and real-time PCR for blood or urine samples. CMV IgM and CMV IgG were tested using an ELISA kit according to the manufacturer's instructions (DiaSorin S.p.A., Italy). For testing CMV in urine, urine samples were collected and cultured using the shell vial culture method (Chemicon, Temecula, CA, USA). According to the manufacturer's instructions (Daan Gene Company of Zhongshan University, China), fluorescence quantitative CMV-DNA kit was used to quantify CMV-DNA. DNA level > 103 copies/ml indicated replication, which was considered positive in this study. CMV gB, gN and gH genotype analysis were done by nPCR and RFLP as reported [[11], [13]].

Statistical analysis was conducted using the SPSS ver. 21.0 software (SPSS, Inc., Chicago, IL, USA). Genotype distribution among congenitally and perinatal infected patients, the relationship between the gB, gN, and gH genotypes and the outcome of CMV infections were analyzed using chi-square test for ratio comparison. Logistic regression analysis was used to assess the associated risk between particular genotypes and the variables of the study. A P-value less than 0.05 was considered to be statistically significant.

25 immunocompetent patients with CAP were analyzed, including 7 congenital infections and 18 perinatal infections. Hepatobiliary symptoms were noted in 9 (36.0%,9/25) patients, including jaundice (24.0%,6/25), hepatitis (8.0%,2/25), and cholestasis (4.0%,1/25). Other clinical symptoms including respiratory symptoms (48.0%,12/25), anemia (24.0%,6/25), neutropenia (20.0%,5/25), intracranial hemorrhage (8.0%,2/25), and sepsis-like (8.0%,2/25) are presented in Table 1.

Table 1. The baseline characteristics and distribution of CMV genotypes among CMV-associated thrombocytopenia and asymptomatic patients.

1 ICH: Intracranial hemorrhage.

The genotypes were successfully amplified in 24 of 25 cases, and PCR amplification was unsuccessful in 1 of 25 cases. The distribution of gB genotypes in this present study was gB1 (70.8%,17/24), gB2 (12.5%,3/24), and gB3 (16.7%,4/24). No gB4 genotype was found. Greater prevalence of gB1 (75.0%,15/20) in moderate to severe CMV disease was noted (χ2 = 8.568, p = 0.014) (Table 2). Compared with the genotype distribution in CMV infected children in previous studies, there were no differences in the distribution of gB genotypes in the infants infected with CAP (Table 3).

Table 2. Distribution of CMV genotypes in different severity of CMV disease.

Table 3. Distribution of CMV genotypes among infants with different clinical manifestations.

The overall distribution of gN genotypes in this study cohort was as follows: gN1(12.5%,3/24), gN2 (29.2%,7/24), gN3 (12.5%,3/24) and gN4 (45.8%,11/24). Comparing results with asymptomatic and mildly CMV infection patients, the gN4 and gN2 were the most prevalent genomic variants in CAP patients (χ2 = 14.198, p = 0.003).

The gH1, gH2 and gH1/gH2 genotypes were distributed in 41.7% (10/24), 54.2% (13/24) and 4.2% (1/24) of the patients, respectively. The gH1 genotype occurred more frequently in CMV infection and other organ/system disorders than gH2, whereas the opposite tendency was observed in the CAP cases, where the gH2 genotype was predominant.

In the logistic regression analysis, the gH2 (p = 0.031) genotype was associated with an elevated risk of developing thrombocytopenia. Conversely, the gB2 (p = 0.020), gN1 (p = 0.018) and gN3 (p = 0.008) genotypes were associated with a reduced risk of thrombocytopenia. In addition, gB1 (p = 0.033) and gN2 (p = 0.038) represented the most virulent genotypes and were associated with severe manifestations (Table 4).

Table 4. Genotypes association with CMV-associated thrombocytopenia.

Some previous studies showed that the most prevalent genotype in Chinese children was gB1 and the rate of gB1 in infants with CAP was higher than infants with other organ/system disorders or asymptomatic infants [[9], [11], [14], [16]]. Compared with the genotype distribution in CMV infected children in previous studies, there were no significant differences in the distribution of gB genotype in the infants infected with CAP. For this reason, we consider it was not appropriate to conclude that there was an association between this genotype of CMV and CAP. A potential relation between gB genotypes and outcome was observed following comparison of severity of CMV disease in control group, which displayed a prevalence of the gB1 variants in associations with more severe CMV disease. This implied that the gB1 genotype was more virulent in infants, responsible for symptomatic CMV disease. On the contrary, the CMV strain with gB2 genotype may represent a less virulent virus phenotype, especially considering that the variation is a typical AD169-like glycoprotein, which is far away from CMV clinical isolates in immunology [[17]]. In the logistic regression analysis, the gB2 genotype was associated with a reduced risk of thrombocytopenia, suggesting that gB2 genotype may have a lower blood cell tropism in infants with CMV infection.

Minimal data are available on the the possible relationship between specific gN genotypes and CAP during early infancy, especially in congenitally and perinatal infected infants. This is the first report on gN genotypes of CMV clinical strains and their potential correlation with CAP. In our study, the gN4 genotype was most prevalent in infants with CAP, followed by genotype gN2, while these genotypes were infrequent in asymptomatic or mildly symptomatic infants with CMV infection. Previous studies have demonstrated that, compared with CMV gN1 and gN3, gN4 represents the most virulent genotype and is associated with severe manifestations [[18], [20]]. Our results confirmed that the gN1 and gN3 variants are associated with less severe disease and suggest that both gN1 and gN3 genotypes of CMV might have a decreased risk of thrombocytopenia in infected infants. In addition, the gN2 genotype was detected in 35.0% (7/20) of infants with severe manifestations and was associated with at least a 7-fold increased risk of developing more severe CMV disease. Compared with the genotype distribution in CMV infected infants in previous study [[21]], thrombocytopenia occurred more frequently in infants infected with the CMV gN2 genotype, although the proportion of this genotype was less than that of gN4 in CAP infants. Further study of these differences is required.

In the reported cases, the gH1 genotype occurred more frequently in Chinese children with acquired CMV infection than gH2 genotype [[12], [22]]. However, the opposite tendency was observed in the CAP cases in our study, where the gH2 genotype was predominant. Our results showed that the gH2 genotype was associated with at least a 7-fold increased risk of developing CAP. All these results suggested that the gH2 genotype might be associated with CAP in infants. Besides, Nahar S et al. [[6]] have described that the gH2 variant of CMV was significantly more prevalent than the gH1 variant in ulcerative colitis active patients receiving immunosuppressive drugs, which possibly indicates weaker virulence. This is similar to another report in sensorineural hearing loss patients [[24]]. However, our findings showed otherwise. No genotypes of gH had been confirmed to be related to virulence. This might be due to different study population, clinical manifestations, or, as in our study, a relatively small sample size, leading to failure to detect a small difference in virulence between the gH genotypes if one existed.

There are few proven risk factors regarding outcome prediction or response to treatment and little progress has been made on CAP in congenital and perinatal infected infants. If specific genotypes do have a higher prevalence in CMV infected patients, the knowledge would be helpful in predicting the possible relationship between genotypes and specific clinical manifestations or severity of CMV disease and developing preventive measures. Several general points can be made on the basis of these cases. First, the specific genotype associations should be analyzed in larger populations in a prospective study with detailed clinical data available for each patient to determine whether specific genomic variants can provide additional prognostic or predictive information on CMV disease progression. Multi-centric studies with larger sample size of infants from diverse backgrounds may provide more information. Second, the potential significant differences in genotype distribution found in different studies may be based on geographical distribution, study population or clinical manifestations [[25]]. Finally, the lack of statistical significance may reflect a limited number of the subjects enrolled.

No potential conflict of interest was reported by the author(s).