Maja Cupic

Prevalence of hepatitis B virus MHR mutations and their correlation with genotypes and antiviral therapy in chronically infected patients in Serbia

Understanding the prevalence and diversity of HBsAg variants in a population is fundamental to assay design and planning vaccination programs. It has been shown that mutations within the S gene, caused by selection or natural variation, can lead to false‐negative results in assays for HBsAg, or have clinical implications, such as evading anti‐HBV immunoglobulin therapy or vaccine‐induced immunity. The region of HBsAg where most of these mutations occur is known as the major hydrophilic region (MHR). The aim of this study was to determine the prevalence and mutational patterns of MHR mutations in patients with chronic hepatitis B, and their correlation with patient characteristics, viral factors and antiviral therapy. The study comprised 164 plasma samples from patients with chronic hepatitis B, of which, 34.8% were on long‐term lamivudine monotherapy. Direct sequencing of part of the S/pol gene was used for identification of HBsAg mutations, HBV genotypes, subgenotypes and HBsAg subtypes. The overall frequency of MHR mutations was 22.6%, but it varied significantly between untreated and treated patients (16.8% vs. 33.3%). The most frequent substitution was at position 120 (9.1%) whereas the most common vaccine‐escape position, 145, was affected in 1.8% of isolates. The presence of MHR mutations was correlated with genotype D, subgenotype D3, and ayw2/ayw3 HBsAg subtypes and to older age (>40 years). It is concluded that natural viral variability present in a geographical region, duration of infection, and antiviral therapy are among the major factors associated with the occurrence of MHR mutations. J. Med. Virol. 82: 1160–1167, 2010.

Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients.

Seven strains of Epstein–Barr virus (EBV) are defined based on C‐terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30‐bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C‐terminal region of LMP1 in Serbian isolates. This study included 53 EBV‐DNA‐positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506‐bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non‐deleted (66%), and the rest had 30‐bp, rare 69‐bp, or yet unknown 27‐bp deletions, which were not related to malignant or non‐malignant isolate origin. However, the majority of 69‐bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33‐bp repeats were found in the majority of non‐deleted isolates (68.6%), whereas most 69‐bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95‐8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical‐associated characteristics in the LMP1 C terminus of investigated isolates. J. Med. Virol. 84:632–642, 2012.

Multiple sclerosis: individualized disease susceptibility and therapy response

Multiple sclerosis (MS) is a heterogeneous disease in which diverse genetic, pathological and clinical backgrounds lead to variable therapy response. Accordingly, MS care should be tailored to address disease traits unique to each person. At the core of personalized management is the emergence of new knowledge, enabling optimized treatment and disease-modifying therapies. This overview analyzes the promise of genetic and nongenetic biomarkers in advancing decision-making algorithms to assist diagnosis or in predicting the disease course and therapy response in any given MS patient.

The influence of single and combined IL28B polymorphisms on response to treatment of chronic hepatitis C

BACKGROUND Three single nucleotide polymorphisms (SNPs) near IL28B gene were shown to be highly predictive of sustained virological response (SVR) in patients with chronic hepatitis C virus (HCV) infection. OBJECTIVES This study attempted to demonstrate the role of single and combined IL28B polymorphisms (rs8099917, rs12979860 and rs12980275) and other host and viral factors in predicting response to treatment, in Caucasian patients infected with HCV genotype 1. STUDY DESIGN The IL28B genotypes at 3 SNPs were determined in 106 patients who underwent standard 48-week therapy and out of which 55.7% achieved SVR. RESULTS Patients carrying genotypes CCrs12979860 or AArs12980275 were 3.5 and 3 times more likely to achieve SVR, respectively. Genotypes GGrs8099917 and TTrs12979860 were identified as predictors of treatment failure. The presence of IL28B profiles including at least one of the favourable genotypes was identified as the most important factor associated with SVR, followed by younger age and lower grade of histological activity. Of all patients who achieved SVR, 88.1% was carrying one of these IL28B profiles. The strongest PPV of single SNPs for achieving SVR was observed for CCrs12979860 (76.9%). The presence of GGrs8099917 showed the strongest NPV of 85.7%. The correlation of SNPs with other host and viral factors revealed association of TTrs8099917 and lower AST levels. CONCLUSIONS Results of this study confirm that all investigated IL28B polymorphisms are associated with treatment response and that presence of any of the favourable IL28B genotypes can be considered independent pretreatment determinant of the effectiveness of therapy.

HHV-8-related hemophagocytic lymphohistiocytosis in a boy with XLP phenotype.

We report a 2.5-year-old boy with an X-linked lymphoproliferative disease (XLP) phenotype who presented with human herpes virus-8 (HHV-8)-related hemophagocytic lymphohistiocytosis (HLH). XLP is a rare primary immunodeficiency characterized by extreme susceptibility to herpes viruses, mainly Epstein-Barr virus (EBV). Approximately 60% of patients with XLP present with fulminant mononucleosis associated with HLH, whereas remaining patients present with hypogammaglobulinemia or lymphoproliferative disease. Most commonly, one of the XLP phenotypes appears after exposure to EBV, but at least 12% of affected individuals developed symptoms without an evidence of EBV infection. Rarely, patients with XLP may present with central nervous system vasculitis or aplastic anemia. HHV-8 is lymphotrophic and it is associated with lymphoproliferative disorders and Kaposi sarcoma in immunodeficient hosts. Kaposi sarcoma rarely occurs in children with well-defined primary immunodeficiency. Also, HHV-8-related HLH was previously reported in 2 siblings with a perforin gene deficiency. Recently, it became evident that besides EBV, other viruses may trigger the symptoms in XLP. We report for the first time HHV-8-related HLH in EBV-negative pediatric patient with an XLP phenotype.

Distribution of JC Virus Genotypes Among Serbian Patients Infected With HIV and in Healthy Donors

Certain factors lead to increased reactivation of JC virus (JCV) and immunodeficiency seems to be the most important. JCV isolates can be classified into eight different genotypes and several subtypes based on nucleotide difference in the VP1 gene. JCV genotypes are strongly associated with particular ethnic groups and frequently used as genetic markers for human evolution and migration. The aim of this study was to determine the frequency of JCV urinary shedding and genotype distribution in Serbia among patients infected with HIV and healthy donors. Urine samples from 107 healthy donors and 93 patients infected with HIV were collected. PCR followed by sequence analysis was carried out using primers specific for VP1 and NCRR of the virus genome. Excretion rate of JCV‐DNA in urine was higher in patients infected with HIV than in healthy donors (44.1% vs. 31.7%) although statistical significance was not found. Within the group infected with HIV, the degree of immunosuppression (measured by CD4+ cell count) did not influence JCV excretion rate. Sequence analysis of JCV NCRR from both patients infected with HIV and healthy donors showed a pattern identical to archetype structure. In healthy Serbian donors the predominant genotype was 1 (41.2%), followed by 4 (32.4%) and 2 (26.4%). On the other hand, genotype distribution pattern was different in patients infected with HIV: 2 (43.9%), 1 (31.7%), and 4 (24.4%). This study showed that European, Eurasian, and Indian types are circulating in Serbia and that distribution corresponds to the origin of the inhabitants of Serbia. J. Med. Virol. 86:411–418, 2014.

Characterization of the Variability of Epstein-Barr Virus Genes in Nasopharyngeal Biopsies: Potential Predictors for Carcinoma Progression.

Epstein-Barr virus (EBV) infection is a significant factor in the pathogenesis of nasopharyngeal carcinoma, especially in the undifferentiated carcinoma of nasopharyngeal type (UCNT, World Health Organization type III), which is the dominant histopathological type in high-risk areas. The major EBV oncogene is latent membrane protein 1 (LMP1). LMP1 gene shows variability with different tumorigenic and immunogenic potentials. EBV nuclear antigen 1 (EBNA1) regulates progression of EBV-related tumors; however, the influence of EBNA1 sequence variability on tumor pathogenesis is controversial. The aims of this study were to characterize polymorphisms of EBV genes in non-endemic nasopharyngeal carcinoma biopsies and to investigate potential sequence patterns that correlate with the clinical presentation of nasopharyngeal carcinoma. In total, 116 tumor biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), collected from 2008 to 2014, were evaluated in this study. The genes EBNA2, LMP1, and EBNA1 were amplified using nested-PCR. EBNA2 genotyping was performed by visualization of PCR products using gel electrophoresis. Investigation of LMP1 and EBNA1 included sequence, phylogenetic, and statistical analyses. The presence of EBV DNA was significantly distributed between TNM stages. LMP1 variability showed six variants, with the detection of the first China1 and North Carolina variants in European nasopharyngeal carcinoma biopsies. Newly discovered variants Srb1 and Srb2 were UCNT-specific LMP1 polymorphisms. The B95-8 and North Carolina variants are possible predictors for favorable TNM stages. In contrast, deletions in LMP1 are possible risk factors for the most disfavorable TNM stage, independent of EBNA2 or EBNA1 variability. A newly discovered EBNA1 subvariant, P-thr-sv-5, could be a potential diagnostic marker, as it represented a UCNT-specific EBNA1 subvariant. A particular combination of EBNA2, LMP1, and EBNA1 polymorphisms, type 1/Med/P-thr was identified as a possible risk factor for TNM stage IVB or progression to the N3 stage.

Oncogenic viruses and their role in tumor formation

Oncogenic viruses trigger persistent infections, which can stimulate uncontrolled cell growth by inducing cell transformation. Different oncogenic viruses use different mechanisms for infecting cells. Most oncogenic DNA viruses integrate transforming sets of genes into the host chromosome and encode proteins that bind and inactivate cell growth regulatory proteins, such as p53 and retinoblastoma gene product. Tumourous RNA viruses use different oncogenic mechanisms. Some of them encode oncogenic proteins that are almost identical to the cellular proteins involved in the control of cellular growth. The overproduction or altered function of these oncogenic materials stimulates cell growth. These RNA viruses can cause tumours rapidly. The second group of oncoviruses integrates their promoter sequences and viral enhancers near to the cellular growth-stimulating gene, initiating the transformation of the cell. The third group of RNA tumour viruses encodes a protein tax that transactivates the expression of cellular genes. Virus-induced malignant transformation of the cell represents the first step in the complex process of oncogenesis.

The sequence analysis of Epstein–Barr virus EBNA1 gene: could viral screening markers for nasopharyngeal carcinoma be identified?

Epstein–Barr virus (EBV) has been identified as a group 1 carcinogenic agent, particularly for nasopharyngeal carcinoma (NPC). The sequence diversity of EBV nuclear antigen 1 (EBNA1) reflects region-restricted polymorphisms, which may be associated with the development of certain malignancies. The aims of the present study were to evaluate EBV EBNA1 gene polymorphisms circulating in NPC, infectious mononucleosis, and isolates from patients with transplanted organs to determine if EBNA1 sequence specificities are useful as viral biomarkers for NPC. Forty biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), 31 plasma samples from patients with mononucleosis syndrome, and 16 plasma samples from patients after renal transplantation were tested in this study. The EBNA1 gene was amplified by nested PCR. Further investigation included sequencing, phylogenetic, and statistical evaluations. Eighty-seven sequences were identified as one of the four EBNA1 subtypes, P-Ala, P-Thr, V-Val, and V-Ala, with further classification into ten subvariants. Of these, P-Thr-sv-1 and P-Thr-sv-3 have never been identified in Europe, while V-Val-sv-1 was newly discovered. Statistical analysis revealed significant differences in the distribution of EBNA1 P-Thr subvariants between the three groups of patients, with noticeable clustering of P-Thr-sv-5 in NPC isolates (p < 0.001). EBV EBNA1 showed no sequence specificity in primary infection. This research revealed a newly discovered EBNA1 subvariant. Importantly, EBNA1 P-Thr-sv-5 showed carcinoma-specific EBNA1 variability. Thus, identification of this subvariant should be considered as a viral screening marker for NPC or UCNT.

Analysis of variability of urinary excreted JC virus strains in patients infected with HIV and healthy donors

In immunocompromised individuals, especially in patients with T cell immunodeficiency, reactivation of JCPyV can cause serious life-threatening diseases. Nowadays, HIV infection is one of the most important factor for reactivation of JCPyV and the development of of the progressive multifocal leukoencephalopathy (PML). Mutations in the outer loops of the VP1 region can lead to the selection of the viral variants with changed tropism and increased pathological potential. The aims of this study were to determine sequence variation and amino acid changes within VP1 loops and the structure of non-coding control region (NCCR) of urinary excreted JCPyV isolates among HIV-infected patients and healthy donors. Single urine samples from 114 HIV-infected patients and 120 healthy donors were collected. PCR was performed for amplification of VP1 and NCCR. Amplified fragments were directly sequenced and analyzed by using bioinformatics tools. Nucleotide substitutions were detected within DE and EF loops and in the β-sheets of both studied groups. In HIV-infected patients group, 70% of mutations were detected within receptor domains. Among healthy donors, one mutation was identified within β-sheets while the remaining were located within receptor domains. The most prevalent mutation was L157V in both groups. Analysis of NCCR revealed that all isolates had archetype structure with some minor changes. Since single point mutations at specific place within outer loop of VP1 region can cause formation of variants with changed receptor specificity, identification of these mutations in HIV-infected patients can help to single out those with higher risk for development of polyomavirus-associated diseases.