Journal of Medical Virology 87:1737–1748 (2015)

Human Cytomegalovirus UL55, UL144, and US28 Genotype Distribution in Infants Infected Congenitally or Postnatally Edyta Paradowska,1* Mirosława Studzi
nska,1 Patrycja Suski,1 Beata Kasztelewicz,2 3
Małgorzata Wisniewska-Ligier, Barbara Zawili
nska,4 Zuzanna Gaj,5 and Dorota Nowakowska5,6 1

Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland 2 Department of Clinical Microbiology and Immunology, The Children’s Memorial Health Institute, Warsaw, Poland 3 3rd Department of Pediatrics, Polish Mother’s Memorial Hospital Research Institute, Lodz, Poland 4 Department of Virology, Jagiellonian University Medical College, Cracow, Poland 5 Department of Fetal-Maternal Medicine and Gynaecology, Polish Mother’s Memorial Hospital Research Institute, Lodz, Poland 6 Department of Fetal-Maternal Medicine and Gynaecology, 3rd Chair of Gynaecology and Obstetrics, Medical University, Lodz, Poland

Cytomegalovirus (CMV) is the most common cause of congenital infection. This pathogen exhibits extensive genetic variability in the genes that encode structural envelope glycoproteins, regulatory proteins, and proteins that contribute to immune evasion. However, the role of specific viral strains in the outcome of congenital CMV infection is unclear. Variation in the UL55 gene encoding glycoprotein B (gB), the UL144 gene encoding TNF a-like receptor, and the US28 gene encoding b-chemokine receptor was determined in 60 newborn infants with congenital CMV infection and 90 infants with postnatal or undefined CMV infection. CMV polymorphisms were studied in relation to disease outcome and viral load. Genotyping was performed by a sequencing analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. The results demonstrated that (1) the UL55 and US28 genotype distributions were similar among the group of congenital and postnatal CMV infection; (2) the UL144 B1 genotype was more prevalent in congenital than in postnatal infection and was detected in 70% of newborns with asymptomatic congenital infection; and (3) none of the examined genotype was significantly linked with symptomatic CMV infection. No relationship was observed between genotype and viral load. The results revealed that UL55, UL144, and US28 polymorphisms are not associated with the outcome of CMV infection in infants, but the presence of UL144 B1 genotype might be virological marker of asymptomatic infection at birth. J. Med. Virol. 87:1737– 1748, 2015. # 2015 Wiley Periodicals, Inc. C 2015 WILEY PERIODICALS, INC. 

KEY WORDS:

cytomegalovirus (CMV); congenital and postnatal infection; glycoprotein B (gB); gene polymorphism; UL144; US28

INTRODUCTION Human cytomegalovirus (CMV) is the leading cause of intrauterine viral infection, affecting 0.3–0.7% of live births in industrialized countries [Griffiths 2002; Dollard et al., 2007; Kenneson and Cannon, 2007] and 1–5% in developing countries [Stagno et al., 1982; van der Sande et al., 2007; Mussi-Pinhata et al., 2009; Manicklal et al., 2013]. CMV may cause pregnancy complications such as intrauterine growth restriction and birth defects. Congenital CMV (cCMV) infection may result in spontaneous abortion, stillbirth, microcephaly, hepatosplenomegaly, jaundice, petechiae, or chorioretinitis

Grant sponsor: European Regional Development Fund under the Operational Programme Innovative Economy; Grant number: POIG.01.01.02-10-107/09; Grant sponsor: Island, Liechtenstein and Norway through the EEA Financial Mechanism, and the Polish national budget funds for research and science; Grant number: PL0270. Conflict of interest: The authors declare that there are no conflicts of interest.
Correspondence to: Edyta Paradowska; Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland. E-mail: [email protected] Accepted 26 March 2015 DOI 10.1002/jmv.24222 Published online 29 April 2015 in Wiley Online Library (wileyonlinelibrary.com).

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[Pass et al., 2006; Adler et al., 2007]. Approximately 10% of newborns infected congenitally are symptomatic at birth [Kenneson and Cannon, 2007]; if symptoms of CMV infection are present, they are frequently non-specific for CMV infection. When disabilities, such as hearing loss, mental retardation, or visual impairment, become apparent in next months or years, it is generally too late to identify cCMV infection as the culprit [Cannon 2009]. Both host and viral factors may affect the outcome of infection. CMV virulence may depend on genetic variability in several regions of the genome. The CMV genome, the largest of all human herpesviruses, is a linear 235–240 kbp double-stranded DNA and contains more than 200 open reading frames (ORFs) [Murphy et al., 2003; Dolan et al., 2004]. Different CMV genes encoding envelope glycoproteins (e.g., gB, UL55; gN, UL73; gH, UL75) and homologs of cytokines/chemokines/chemokine receptors, including UL144 and US28, have been studied due to their potential relevance for cell tropism and virulence [Barbi et al., 2001; Pignatelli et al., 2004, 2010; Paradowska et al., 2012, 2013, 2014a,b; Pati et al., 2013]. CMV gB is a major component of the virion envelope, and it acts as a fusion protein [Wille et al., 2013]. Based on the nucleotide sequence encoding the variable region, CMV strains have been classified into four main variants, gB1-4, or into the rare variants gB5-7 [Trincado et al., 2000]. A genotyping analysis showed that the gB genotype distribution differs with geographic origin. Different gB genotypes can cause congenital CMV infection, but none of these genotypes appears to be associated with CMV disease [Bale et al., 2000; Barbi et al., 2001; Arista et al., 2003; Picone et al., 2004]. The UL144 gene, located in the UL/b’ region, encodes a homolog of the herpes simplex virus entry mediator (HVEM), which is a member of the tumor necrosis factor receptor superfamily (TNFRSF) [Benedict et al., 1999]. The UL144 is a membrane-anchored glycoprotein retained intracellularly. It consists of two cysteine-rich domains (CRDs), membrane extension region, transmembrane domain, and a short cytoplasmic tail. The UL144 protein lacks an additional CRD3 region and does not bind any known TNF-related ligands [Benedict et al., 1999]. In fact, only B and T lymphocyte attenuator (BTLA) has been shown to bind to the UL144 protein [Cheung et al., 2005]. It may inhibit T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM. UL144 can also activate a transcription factor, TNFR-activated factor 6 (TRAF6), leading to NF-kB activation and chemokine CCL22 expression [Poole et al., 2006], a chemoattractant of regulatory T-cells and Th2 cells. A phylogenetic analysis shows that the nucleotide sequences of the UL144 gene cluster into five major groups: A, B, C, AB, and AC [Lurain et al., 1999; AravBoger et al., 2002]. Arav-Boger et al. [2002, 2006] has suggested that polymorphisms in the UL144 gene may be associated with cCMV disease. In contrast, no J. Med. Virol. DOI 10.1002/jmv

Paradowska et al.

significant relationship between UL144 genotypes and disease was found in other studies [Lurain et al., 1999; Bale et al., 2001; Picone et al., 2005; Tanaka et al., 2005; Mao et al., 2007; Heo et al., 2008; Yan et al., 2008; Paradowska et al., 2012; Nijman et al., 2014]. The US28 ORF encodes a homolog of cellular G protein-coupled receptor (GPCR), a functional b-chemokine receptor that binds and sequesters extracellular chemokines and modulates host immune responses [Pleskoff et al., 1997; Streblow et al., 1999]. US28 can bind CC chemokines to induce increases in intracellular calcium levels and the migration of infected cells [Billstrom et al., 1998; Streblow et al., 1999]. It also appears to impact cell-to-cell spread of the virus [Noriega et al., 2014]. The US28 variability is located mainly at the two ends of the gene. To establish which genotypes are associated with congenital and postnatal CMV infections in infants, the UL55, UL144, and US28 sequences obtained from 150 infants were amplified and sequenced. We attempted to explore whether specific clades are associated with symptomatic cases of CMV infection and the association between genotype and viral load. The previous results from 25 newborns with cCMV infection were included in this study [Paradowska et al., 2012]. (This research was presented in part at the 36th Annual International Herpesvirus Workshop, Poland, Gdansk, 24–26 July 2011, abstract 8.17.) MATERIALS AND METHODS Between February 2008 and March 2011, blood, urine and/or cerebrospinal fluid (CSF) samples were obtained from selected 150 infants infected with CMV. The specimens studied consisted of 76 blood, 110 urine, and 3 CSF samples. In all examined children, CMV infection was confirmed on the basis of CMV DNA detection and the results of serological assays. A total of 69 samples/isolates were obtained from 60 newborns with cCMV infection, and 120 samples/isolates were obtained from 90 infants with postnatal or undefined CMV infection (pCMV). The CMV isolates from infected infants were obtained from Central Poland: Department of Clinical Microbiology and Immunology, the Children’s Memorial Health Institute in Warsaw (64 cases), 3rd Department of Paediatrics (37 cases) and Department of Fetal-Maternal Medicine and Gynaecology (25 cases), the Research Institute of the Polish Mother’s Memorial Hospital in Lodz, and from Southern Poland–the Jagiellonian University Collegium Medicum, Cracow (24 cases). All children were classified as having symptomatic or asymptomatic infection on the basis of physical, instrumental, and laboratory findings. Infants were classified as having symptomatic infection when they had any of the clinical findings suggestive of CMV infection, including, for example, intrauterine growth retardation, jaundice, petechiae, hepatosplenomegaly, purpura, microcephaly, intracerebral calcifications, sensorineural hearing loss, neurological dysfunction, and others.

CMV gB, UL144, and US28 Genotypes in Infants

Plasma was obtained by centrifugation of peripheral blood for 20 min at 1,600g. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation in a Ficoll-Paque PLUS (Amersham Biosciences AB, Uppsala, Sweden) gradient. The protocol and study documents were approved by the appropriate Ethics Committees, and written informed consent was obtained from the parents of the children who participated in the study. Total DNA was extracted with the QIAamp Blood DNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. DNA was extracted from 200 ml of blood, urine, CSF, and 5  105 PBMCs, eluted in 100 ml of elution buffer, and then stored at 20˚C. The CMV genes were amplified with Taq DNA polymerase (Fermentas, Glen Burnie, MD) using a nested PCR method, as described previously [Paradowska et al., 2012]. DNA isolates were examined with forward 50 -CGCGGCAATCGGTTTGTTGT-30 and reverse 50 -CGAGAAGAATGTCACCTGCC-30 primers flanking the variable region of the UL55 gene (82810–83410) as a first round of PCR amplification. When the product amplified by the outside primers was either weak or negative, a second round of PCR using the nested forward primer 50 -TCCGAAGCCGAAGACTCGTA-30 and reverse primer 50 -GATGTAACCGCGCAACGTGT-30 and the product from the first round was carried out to detect the amplicon (82942–83352) [Lurain et al., 1999]. For detection of the UL144 gene, the product (181217–181942) was amplified with the primer pair forward, 50 -AACCGCGGAGAGGATGATAC-30 , and reverse, 50 -ACTCAGACACGGTTCCGTAA-30 ; the nested amplicon (181308–181921) was amplified with primers forward, 50 -GTTCGGCCCCATGAGTTATT-30 , and reverse, 50 -GTGTGACTTCATCGTACCGT-30 [Stranska et al., 2006]. The hypervariable fragment of the US28 gene was amplified using specific sets of primers: forward 50 -GTGAACCGCTCATATAGACC-30 and reverse 50 -GAAACAGGCAGTGAGTAACG-30 (220036– 220464). A heminested PCR was performed with the following reverse primer 50 -CATCCACAGAGGTAGTGTAC-30 and forward primer 50 -GTGAACCGCTCATATAGACC-30 (220036–220401) [Arav-Boger et al., 2002]. The conditions for amplification with all primers sets were 94˚C for 2 min, 30 cycles at 94˚C for 30 sec, 55˚C for 30 sec, and 72˚C for 30 sec, and 72˚C for 4 min. DNA isolates from MRC-5 cells (ATCC CCL-171, American Type Culture Collection, Rockville, MD) infected with CMV laboratory strain AD-169 (ATCC VR-538), Towne (ATCC VR-977), and Davis (ATCC VR-807) were used as a positive control, and nuclease-free water was used as a negative control. Positive and negative controls were included with each run. All amplifications were carried out with a Veriti 96 Well Thermal Cycler (Applied Biosystems, Foster City, CA). The amplicons were analyzed using 1.2% agarose gel electrophoresis and UV visualization (FluorChem8800, AlphaInnotech). The PCR products were sequenced by the DNA synthesis service Genomed (Warsaw, Poland) using

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the MiSeq system (Illumina, San Diego, CA). The obtained sequences were analyzed and verified with the Chromas-Win95/98/NT/2000/XP and Basic Local Alignment Search Tool (BLAST). The results were compared with reference sequences in the GenBank database. The CLC Protein Workbench v. 5.4 for alignment and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) for phylogenetic tree construction were used. The viral loads in samples were measured by quantitative real-time PCR, as described previously [Paradowska et al., 2006, 2012]. To amplify CMV DNA, primers and probe were defined for the UL55 gene. The TaqMan probe was labeled at the 50 end with FAM (6-carboxyfluoroscein) and at the 30 end with TAMRA (6-carboxytetramethylrhodamine) [Hassan-Walker et al., 2001; Temperton et al., 2003]. Real-time PCR was carried out using a 7900HT Fast Real-Time PCR System (Applied Biosystems). The data were analyzed using Statistica 8.0 and SPSS 17.0 software. Differences in the distributions of CMV genotypes and the association between the genotype and outcome of CMV infection were analyzed using Fisher’s exact test. Logistic regression models were used to evaluate the association between the virus genotype and the symptomatic infection. To compare the viral load between the various groups, a unpaired Student’s t test was performed. Only P values of 0.05). The gB2 genotype was prevalent in the congenital infections from Central Poland, whereas the gB1 and gB2 genotypes were distributed at similar frequencies in South Poland (data not presented). An amino acids sequence alignment of the UL55 and UL144 products (A) and a phylogenetic tree constructed using these sequences (B) is shown in Figures 1 and 2. If different clinical samples from one patient was obtained, it was possible to detect mixed infection. In almost all cases of cCMV infection, only one sample was investigated, and only one virus strain could be detected. Infection with mixed gB genotypes was detected in six infants with pCMV infection (Table I). The obtained UL144 gene sequences were clustered into four main genotypes according to the J. Med. Virol. DOI 10.1002/jmv

nomenclature of Arav-Boger et al. [2002] and Lurain et al. [1999]. The distribution of UL144 genotypes in the groups of infants with cCMV was as follows: 31.7, 61.7, 3.3, and 5.0% for genotypes A, B, C, and AB, respectively (Table II, Fig. 2). Genotypes A (37.8%), B (66.7%), C (12.2%), and AB (2.2%) were common in the infants with pCMV infection. The AC genotype was not detected in any child. In addition, recombinant A1/AB genomic variant was identified in one infant with pCMV infection. Mixed infection with two strains was found in one patient with cCMV infection (1.7%), whereas two or three different genomic variants were found in 18/90 (20%) of the infants infected postnatally. The frequency of the B1 genotype was higher in congenital infection compared to postnatal infection (P ¼ 0.018), whereas the B4 variant was prevalent in postnatal infection (P < 0.001). No other statistically significant association in the UL144 genotype distribution among the infant groups studied was observed.

CMV gB, UL144, and US28 Genotypes in Infants

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TABLE II. Distribution of CMV Genotypes in Infants with Symptomatic and Asymptomatic Infection Prevalence of CMV genotypes in infants, n (%) Congenital infection Gene UL55

Genotype Symptomatic Asymptomatic

gB1 gB2 gB3 gB4 UL144 A1 A2 B1 B2 B3 B4 AB C1 C2 C3 A1/AB US28 A1 A2 B1 D

11 (25.6) 30 (69.8) 2 (4.6) 0 13 (30.2) 4 (9.3) 12 (27.9) 1 (2.3) 4 (9.3) 5 (11.6) 3 (7.0) 1 (2.3) 1 (2.3) 0 0 22 (51.2) 21 (48.8) 0 0

2 (11.8) 15 (88.2) 0 0 2 (11.8) 0 12 (70.6) 0 3 (17.6) 0 0 0 0 0 0 5 (29.4) 12 (70.6) 0 0

Total 13 (21.7) 45 (75.0) 2 (3.3) 0 15 (25.0) 4 (6.7) 24 (40.0)a 1 (1.7) 7 (11.7) 5 (8.3) 3 (5.0) 1 (1.7) 1 (1.7) 0 0 27 (45.0) 33 (55.0) 0 0

Postnatal infection P value Symptomatic Asymptomatic 0.314 0.192 1.000 1.000 0.310 0.570 0.008 1.000 0.393 0.309 0.551 1.000 1.000 1.000 1.000 0.158 0.158 1.000 1.000

21 50 9 1 26 3 16 7 28 2 5 2 4 35 38 3 7

(28.0) (66.7) (12.0) (1.3) (34.7) (4.0) (21.3) 0 (9.3) (37.3) (2.7) (6.7) (2.7) (5.3) 0 (46.7) (50.7) (4.0) (9.3)

4 (26.7) 11 (73.3) 0 0 5 (33.3) 0 4 (26.7) 0 0 5 (33.3) 0 0 0 0 1 6 (40.0) 6 (40.0) 0 3 (20.0)

Total 25 61 9 1 31 3 20

(27.8) (67.8) (10.0) (1.1) (35.6) (3.3) (22.2) 0 7 (7.8) 33 (36.7)b 2 (3.3) 5 (5.6) 2 (2.2) 4 (4.4) 1 (1.1) 41 (45.6) 44 (48.9) 3 (3.3) 10 (11.1)c

P value 1.000 0.766 0.347 1.000 1.000 1.000 0.735 1.000 0.596 1.000 1.000 0.585 1.000 1.000 0.167 0.779 0.575 1.000 0.361

n, number of infants with CMV genotype; Fisher’s exact test was used to compare genotype distribution between the infant groups. a P ¼ 0.018, compared with the UL144 B1 distribution in postnatal/undefined infection. b P < 0.001 (UL144 B4). c P ¼ 0.005 (US28 D), compared with the genotype distribution in congenital infection.

A phylogenetic analysis showed that the US28 sequences from the clinical isolates formed two major A1 and A2 genotypes (Table I). These variant genotypes occurred with similar frequency in the congenital and postnatal infections. There were no sequences related to the B and D genotypes and mixed infection in the group of newborns with cCMV infection. The US28 D genotype was significantly associated with postnatal infection (P ¼ 0.005). The median CMV DNA concentration in urine was significantly higher (1.04  107 copies/ml; range 0– 8.73  108 copies/ml) in newborns with congenital infection than in infants with postnatal CMV infection (2.10  104 copies/ml; range 0–2.21  108 copies/ ml), P ¼ 0.004. The viremia levels ranged from 0 to 8.50  105 copies/ml (median 5.80  103 copies/ml) in newborns and from 0 to 1.46  105 copies/ml (median 3.36  102 copies/ml) in infants (P ¼ 0.031). No relationship between a specific genotype and viral load in clinical samples was observed. The distribution of CMV genotypes in infants with symptomatic or asymptomatic disease was investigated. An analysis of the samples demonstrated that the common gB, UL144, and US28 genotypes were found in both the symptomatic and asymptomatic infant groups (Table II). The UL144 B genotype was associated with both asymptomatic (24/32, 75%) and symptomatic (73/118, 62%) infants. However, the UL144 B1 genotype was detected in 70% (12/17) of newborns with asymptomatic cCMV infection compared with those with symptomatic infection (P ¼ 0.008). The risk of symptomatic CMV infection

was more than fivefold reduced following congenital infection with the UL144 B1 variant (P ¼ 0.006; Table III). Other genotypes were not associated with symptomatic infection (P > 0.05; Table II). However, high percentages of symptomatic infection were detected among infants infected with genotypes A (46/ 53, 86.8%) and C (9/9, 100%). Although different genotypes occurred in both groups of infants with cCMV and pCMV infection, all the mixed infections were found only in the symptomatic patients (Table I). Sequence analysis of the three polymorphic loci revealed that 20/90 (22.2%) of the infants were infected postnatally with two or three CMV strains detected indifferent compartments. Among the group of cCMV infection, only one newborn demonstrated distinct CMV strains in blood and urine samples. DISCUSSION The present study focused on the genotype distribution of the three genes encoding important CMV proteins, UL55, UL144, and US28, in an infant population and the relationship between specific viral genotypes and the clinical outcome of CMV infection. To assess whether CMV polymorphism is crucial for the intrauterine transmission of the pathogen, the frequencies of specific genotypes in two groups of children: with congenital or postnatal CMV infection were compared. The previous study conducted in pregnant women and newborns with congenital infection revealed no correlation between CMV UL55, UL144, and US28 genotypes and the J. Med. Virol. DOI 10.1002/jmv

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Fig. 1. Amino acid alignments of gB from clinical isolates of newborns with congenital CMV infection and the Towne, AD-169, and Davis reference strains. Consensus sequences are shown at the bottom (A). Phylogenetic analysis of the gB in CMV isolates obtained from newborns with congenital CMV infection (B). The reference sequences of laboratory strains Towne, AD-169, and Davis are included for reference. Bold numbers indicate the gB genotype. Whole blood (A), PBMC (B), urine (C), and CSF (D) samples.

disease [Paradowska et al., 2012]. Because the sample population was small, a larger group of patients with congenital infection was selected, and, in addition, a group of infants with postnatal or J. Med. Virol. DOI 10.1002/jmv

undefined CMV infection was included. Both patient groups showed high frequencies of symptomatic CMV infection. Because various genotypes were detected in children infected congenitally, we

CMV gB, UL144, and US28 Genotypes in Infants

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Fig. 2. Amino acid alignments (A) and phylogenetic analysis (B) of the UL144 gene in clinical isolates obtained from newborns with congenital CMV infection. The Towne and Davis were used as reference strains. Abbreviations are the same as those in Figure 1.

J. Med. Virol. DOI 10.1002/jmv

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Paradowska et al.

TABLE III. Assessment of UL144 B1 Genotype as Prognostic Variables for Risk of Symptomatic CMV Infection in Infants Adjusted for CMV DNA copy number Unadjusted Infection Congenital Postnatal

Viremia

Viruria

n (%)

P

OR (95%CI)

P

OR (95%CI)

P

OR (95%CI)

13/60 (21.7) 16/90 (17.8)

0.006 0.651

0.18 (0.05–0.62) 0.75 (0.21–2.66)

0.061 0.832

0.12 (0.01–1.10) 0.76 (0.06–9.73)

— 0.487

— 0.62 (0.17–2.35)

n, number of cases with CMV UL144 B1 genotype among symptomatic patients; OR, odds ratio; 95%CI, 95% confidence interval.

concluded that different virus strains could be transmissible from mother to child. The UL144 B1 genotype was more prevalent in congenital infection and was detected in majority of newborns with asymptomatic congenital infection, whereas the B4 and A1 variant were prevalent in infants with postnatal infection. In addition, no other relationship between genotype and symptomatic infection or virus load was found. CMV uses different strategies to avoid host immune detection and encodes gene products that can modulate the immune response in the host, including TNFa-like receptor and receptor homologs that bind to host chemokines. CMV clinical isolates display genetic polymorphism in several genes, especially in the genomic UL/b’ region that is missing in laboratory isolates [Cha et al., 1996; Prichard et al., 2001; Arav-Boger et al., 2002; Dolan et al., 2004]. The UL144 cytokine receptor gene is located in this region but is present in clinical isolates of CMV. Previous studies have also reported sequence divergence in the US28 gene [Baldanti et al., 1998; Arav-Boger et al., 2002; Goffard et al., 2006]. Hence, it has been suggested that UL144 and US28 variants are good candidates for prognostic markers of congenital infection, which may correlate with CMV disease. The results of the present study show that the UL144 genotype distributions were similar among the strains recovered from infants infected congenitally and postnatally. As different UL144 genotypes could be transmitted vertically from mothers to fetuses, this observation does not appear to have any definite prognostic value [Arav-Boger et al., 2002; Picone et al., 2005; Waters et al., 2010]. It was found that UL144 genotypes B and A were detected generally within the congenital CMV population. As with most of other studies, the UL144C and AB genotypes were the least frequently detected. A similar genotype distribution was observed in other studies [Bale et al., 2001; Waters et al., 2010; Pati et al., 2013; Nijman et al., 2014]. Arav-Boger et al. [2002] and Picone et al. [2005] found a prevalence of genotypes B and C in congenital infections. Infants with postnatal infection were predominantly infected with UL144 B and A genomic variants, whereas the B and C genotypes were the most prevalent among Dutch CMV strains [Nijman et al., 2014]. It was previously observed that the A and C genotypes were associated J. Med. Virol. DOI 10.1002/jmv

with symptomatic outcomes of cCMV infection [AravBoger et al., 2002, 2006; Waters et al., 2010]. Detection of these genotypes was also indicative of serious congenital infection, a high plasma viral load, and were more likely to lead to long-term cCMVassociated clinical manifestations [Waters et al., 2010]. In the present study, the UL144 A and C variants were detected mainly in symptomatic children, although C genotype was found only in a small number of children. There is a report of some groups having a relatively large portion of asymptomatic outcomes caused by UL144 group B (73% of asymptomatic cases) [Heo et al., 2008]. The present study confirmed this observation and provides the demonstration that the UL144 B1 genotype of CMV might be important virological marker of asymptomatic congenital infection. In contrast, Picone et al. [2005, 2007] showed no link between UL144 genotypes recovered from the amniotic fluids and clinical outcomes of cCMV infection. The role of the UL144 variants in the CMV pathogenesis and immune response is unknown. Among CMV clinical strains, the UL144 gene exhibits identity in transmembrane and cytoplasmic domains, whereas high variability in the ectodomain and signal peptide [Lurain et al., 1999]. Despite genetic polymorphism in the CRD1 domain, interaction of UL144 protein with BTLA was similar within all viral genotypes [Cheung et al., 2005]. Poole et al. [2006] have found that the UL144 protein upregulates NF-kB-dependent transcription through a mechanism involving TRAF6 and this activation is concomitant with an increased expression of CCL22. In addition, both A and B genotypes were able to induce NF-kB activation and upregulate chemokine production. However, the sequence diversity in this domain may contribute to variation in reactivity of the specific antibodies to UL144 [Benedict et al., 1999]. The UL55 gene is highly variable in regions corresponding to the N-terminus and the cleavage site between aa 460 and 461 [Chou and Dennison, 1991]. Associations of the gB genotype with the outcome of cCMV infection are controversial [Bale et al., 2000; Trincado et al., 2000; Barbi et al., 2001; Lucacsi et al., 2001; Arista et al., 2003; Yan et al., 2008; Pati et al., 2013], and most studies have revealed no relation between the gB genotype and the outcome of CMV infection. In contrast, some data

CMV gB, UL144, and US28 Genotypes in Infants

indicate that CMV gB1 strains are less virulent than other gB genotypes, whereas gB2 and gB3 are associated with greater virulence in immunocompromised patients. In addition, the gB3 genotype was found to be prevalent in congenital cases with sensorineural hearing loss [Yan et al., 2008]. The distribution of CMV gB genotypes seems to be associated with geographic and/or demographic differences among patients. The gB1 genotype is frequently encountered in European infants infected congenitally [Barbi et al., 2001; Lucacsi et al., 2001; Arista et al., 2003; de Vries et al., 2012; Nijman et al., 2014]. In some studies, both gB1 and gB2 genotypes were dominant in children with cCMV infection [Picone et al., 2004; Rycel et al., 2014]. The dominance of CMV gB2 strains were reported in Mexican infants [Arellano-Galindo et al., 2014], American HIV-infected patients with CMV retinitis [Chern et al., 1998; Drew et al., 2002], and pediatric bone marrow transplant recipients from Japan [Wada et al., 1997]. Our previous results demonstrated that the gB2 genotype was prevalent in cCMV infection and infection in immunocompromised transplant recipients [Paradowska et al., 2012]. In another study, genotype gB1 was prevalent in neonates and infants from Southern Poland [Zawili
nska et al., 2011]. It is suggested that the reasons for such a discrepancy are selection bias and the genotyping method. In this study, symptomatic infection was observed in 71.7% examined newborns and 83.3% infants. It has been observed previously, that the gB2 genotype was more common among infants with symptomatic infections than gB1 genotype [Bale et al., 2000; Barbi et al., 2001]. In addition, the frequency of malformation in infants infected with CMV gB2 genotype was higher than other genotypes [Jin et al., 2007]. The gB2 genotype infected preferentially of T lymphocytes in organ transplant recipients [Meyer-K€onig et al., 1998], caused damage in the nervous system [Tarrag
o et al., 2003], and severe infection in AIDS patients [Bongarts et al., 1996]. Two different methods, PCRsequencing [Paradowska et al., 2012] and real-time PCR [Zawili
nska et al., 2011; Rycel et al., 2014], were used to explore gB variants in Polish pediatric patients. The results of PCR sequencing depend on the original amount of viral DNA and can select among the multiple strains only one potentially present in the original sample, and its strength is as the best approach to avoid mistakes in genotyping. In contrast, several target sequences in a sample can be detected by multiplex real-time PCR including more than one pair of primers at the same time. In the present study, the highest frequency of 75% for the gB2 genotype in cCMV infection was observed. It is suggested that individuals infected with the CMV gB2 genotype had a higher viral load and therefore this genotype was detected more frequently in clinical isolates. Arav-Boger et al. [2002] studied CMV isolates obtained from patients with congenital disease and

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segregated four major genotypes of the CMV US28 protein based on N-terminal mutations. However, it is unclear whether the genetic variability observed among immunocompromised patients reflects the variability of CMV isolates that circulate among CMV-infected children. A comparison of the US28 sequences from AIDS patients with those from children showed that both sequences have their own specific mutation hotspots [He et al., 2011]. Such high-mutation sites from the pediatric isolates were located at the C terminus, whereas those from the AIDS patients were located at the N terminus. Most of the sequences from Chinese infants with suspected congenital CMV infection fell within the G1 group, corresponding to the A1 genotype. Differences in the sequence variability of clinical isolates could affect the migration of leukocytes and lead to different tissue and organ targets [He et al., 2011]. This study confirmed that genotypes A1 and A2 appeared in both symptomatic and asymptomatic newborns, but no significant associations were found between outcome and virologic parameters. A limitation of this study is the relatively small sample size of infants with asymptomatic CMV infection, which may have led to selection bias. The majority of children participating in the study was selected during hospitalization due to severe symptoms. Unless CMV was detected in the urine within 3 weeks of birth, congenital infection could not be distinguished from acquired infection, even when clinical problems suggestive of congenital infection were present. It is possible that the group of infants with postnatal infection may include children with horizontal and vertical CMV transmission. It should be noted that cCMV infections mainly result from recurrent infections among pregnant women, comprising both reactivation of endogenous virus and reinfection with a different strain [Boppana et al., 2001; Yamamoto et al., 2010]. An additional limitation of the study is use of direct PCR-sequencing as genotyping method. Primer sets for a genotypic assays were designed to amplify the variable regions from human CMV genome. To improve PCR efficiency, more sensitive nested or heminested PCR reactions have been optimized. However, PCR method has limitations that result in biases in the template-to-product ratios of the target sequences amplified during PCR. This genotyping method allows detection of the predominant genotype in single specimen and thus, mixed infections could have been missed. As genotypic tests can only detect viral genotypes when these comprise at least 20–25% of the total virus population, the results may be affected by the original amount of viral DNA. The proportion of detected mixed genotypes in infants was lower (20%) in comparison to reports which showed ranges from 31% to 46% [Ross et al., 2011; Zawili
nska et al., 2011; Paradowska et al., 2013; Pati et al., 2013]. Double and triple mixed infections has been reported in at least one-third of J. Med. Virol. DOI 10.1002/jmv

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Paradowska et al.

immunocompromised patients [Manuel et al., 2009; de Vries et al., 2012]. It is possible that mixed infections occurred more commonly in examined infants and they play an important role in the pathogenesis of cCMV infection. The role of mixed infections in disease outcome remains to be defined, although an association of mixed infections with clinical outcome has been demonstrated in transplant patients [Coaquette et al., 2004; PuchhammerSt€ockl et al., 2006]. It should be noted that CMV also evolves within individual infected hosts over time. Earlier studies showed that there are different overall distributions of CMV genotypes in different body compartments [Tarrag
o et al., 2003; Puchhammer-St€ockl et al., 2006; Ross et al., 2011]. Renzette et al. [2013] used high throughput sequencing to sample CMV genomic populations from the urine and plasma of infants with symptomatic cCMV infection at multiple time points during the first year of age. The studies revealed that CMV is generally stable within a host compartment, but rapidly evolves when crossing compartments. The infection and pathogenesis of CMV strain may be defined by the interactions of multiple genes. In the present study, sequence analysis of highly polymorphic regions of CMV genome were investigated in cohort study of Polish infants. To our knowledge, this is the first study on three variable viral genes performed in infants with prenatal and postnatal CMV infection in this part of Europe. An extensive variability in each gene was observed in individual strains; however, there was no correlation between variations. The UL55, UL144, and US28 genotypes do not appear to be prognostic factors in CMV infection in infants. However, the UL144 B1 genotype was associated with a diminished risk of symptomatic cCMV infection. Although most mutations in the CMV genome are stable, the change of only a few can dramatically alter viral fitness or pathogenesis. It is important to characterize the mutations important for the ultimate disease phenotype and host genetic factors that may be responsible for the intrauterine CMV transmission. Future studies using sensitive ultra-deep pyrosequencing technique, with which even low-abundance genotypes at a frequency of less than 1% of the population can be detected [G€orzer et al., 2010], could determine genotypes associated with symptomatic cCMV infection. This knowledge could point out future directions to lessen the burden of CMV disease. ACKNOWLEDGMENTS The authors are grateful to Prof. Teresa Wo
zniakowska-Gęsicka and Prof. Jan Wilczy
nski (Polish Mother’s Memorial Hospital Research Institute, Lodz), Prof. Katarzyna Dzier_zanowska-Fangrat, Dr. Justyna Czech-Kowalska, and Dr. Bo_zena Lipka (The Children’s Memorial Health Institute, Warsaw), Prof. Maria Kornacka (Warsaw Medical University), J. Med. Virol. DOI 10.1002/jmv

Prof. Ryszard Lauterbach, Prof. Jacek J. Pietrzyk, Prof. Magdalena Kosz-Vnenchak, Dr. Dorota Pawlik, and Dr. Tomasz Tomasik (Jagiellonian University Medical College, Cracow) for providing samples from patients. We would like to thank Prof. Zbigniew J. Le
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