Tanja Jovanovic

Maturation of IgG avidity to individual rubella virus structural proteins

BACKGROUNDnthe structural proteins of rubella virus, the capsid protein C and the envelope glycoproteins E1 and E2 were produced in lepidopteran insect cells using baculovirus expression vectors. The C-terminal ends of the corresponding proteins were fused to a polyhistidine tag for easy and gentle purification by metal ion affinity chromatography.nnnOBJECTIVESnto investigate the maturation of natural and vaccinal IgG avidity against individual authentic and recombinant rubella virus (RV) structural proteins.nnnSTUDY DESIGNnthe analysis was carried out using a modified immunoblotting technique where the purified baculovirus-expressed proteins were compared with authentic rubella virus proteins. Altogether, 47 well-characterised serum samples from both naturally infected patients and vaccines were studied.nnnRESULTSnafter natural RV infection, IgG antibodies specific for the E1 protein were predominant not only in terms of levels, but also in terms of rate and magnitude of avidity maturation. The avidity development of the IgG antibodies was much slower in vaccines than in patients after a natural RV infection.nnnCONCLUSIONSntogether, our results indicate that IgG avidity determination in conjunction with immunoblot analysis is useful in the diagnosis of a RV infection. The recombinant proteins showed similar reactivity patterns in the immunoblot analyses as compared with the authentic viral structural proteins, suggesting suitability for serodiagnostics.

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.

Risk factors for hepatocellular carcinoma: a case-control study in Belgrade (Serbia).

Aims and background The objective of this case-control study was to test the existing hypotheses about factors related to the occurrence of hepatocellular carcinoma in the population of Belgrade (Serbia). Methods and study design The investigation was conducted between 2004 and 2007 and consisted of 45 newly diagnosed, histologically confirmed hepatocellular carcinoma patients and 90 individually gender- and age-matched hospital controls. Conditional univariate and multivariate logistic regression analyses were applied. Results A highly statistically significant association (P = 0.001) was demonstrated between hepatocellular carcinoma and HBsAg positivity and the presence of hepatitis C virus antibodies. Diabetes mellitus was significantly (P = 0.018) associated with an increased risk of hepatocellular carcinoma. A statistically significant inverse association was shown between low parity and the risk of hepatocellular carcinoma (P = 0.033). The risk increased significantly with a longer history of cigarette smoking (P = 0.044), as well as the daily consumption of hard liquor (P = 0.049). A weekly intake of fish (P = 0.003) and yogurt (P = 0.003) and daily intake of boiled vegetables (P = 0.001) were reported more frequently by controls than hepatocellular carcinoma cases. In the current study, a high intake of salty food also significantly increased the risk of hepatocellular carcinoma (P = 0.027). Based on multivariate analysis, the presence of hepatitis C virus antibodies (OR = 24.6, P = 0.001) and duration of smoking ≥25 years (OR = 3.8, P = 0.020) were significantly related to hepatocellular carcinoma, whereas the daily consumption of boiled vegetables (OR = 0.1, P = 0.011) was inversely associated with the risk of hepatocellular carcinoma. Conclusions The findings obtained in the current study support the hypotheses that non-viral factors, such as lifestyle factors, reproductive factors, and a history of diabetes, might be involved in the etiology of hepatocellular carcinoma. Free full text available at www.tumorionline.it

Prevalence of oral herpes simplex virus reactivation in cancer patients: a comparison of different techniques of viral detection.

BACKGROUNDnOral reactivation of latent Herpes simplex virus (HSV) infection may easily occur in cancer patients. Virus reactivation can cause oral mucosa damage, worsen already existing lesions caused by stomatotoxic effect of cancer therapy and, whether symptomatic or asymptomatic, ample spreading and promote viral transmission.nnnMETHODSnPolymerase chain reaction (PCR), cell-culture and direct immunofluorescence have been used to determine the frequency of oral HSV reactivation in 60 patients undergoing chemotherapy for different malignancies.nnnRESULTSnBy means of PCR, the presence of viral DNA was detected in 71.7% of patients prior to chemotherapy and in 85.0% after chemotherapy. 33.3% of patients before and 40.0% after chemotherapy were viral-culture positive, while 3.3% of patients before and 11.7% after chemotherapy were positive as shown by direct immunofluorescence. No significant difference in HSV-1 reactivation was found before and after chemotherapy. In addition, no significant difference was found when comparing HSV-1 reactivation in patients with and without mucositis. HSV-2 was not detected in any of the patients.nnnCONCLUSIONSnReactivation of latent HSV is exceptionally frequent in cancer patients. The results of this study suggest that virus reactivation occurs independently of cancer chemotherapy. The potential role of HSV reactivation in oral mucosa damage remains unclear.

HIV-1 subtypes in Yugoslavia.

To gain insight concerning the genetic diversity of HIV-1 viruses associated with the HIV-1 epidemic in Yugoslavia, 45 specimens from HIV-1-infected individuals were classified into subtypes by sequence-based phylogenetic analysis of the polymerase (pol) region of the viral genome. Forty-one of 45 specimens (91.2%) were identified as pol subtype B, 2 of 45 as subtype C (4.4%), 1 of 45 as CRF01_AE (2.2%), and 1 as CRF02_AG recombinant (2.2%). Nucleotide divergence among subtype B sequences was 4.8%. Results of this study show that among HIV-1-infected patients in Yugoslavia subtype B predominates (91.5%), whereas non-B subtypes are present at a low percentage, mostly related to travel abroad.

Disseminated Neonatal Herpes Caused by Herpes Simplex Virus Types 1 and 2

Disseminated neonatal herpes simplex virus (HSV) infection is characterized by progressive multiple organ failure and high mortality rates. It can result from infection with either HSV-1 or HSV-2. We report a case of disseminated neonatal herpes that was caused by HSV-1 and HSV-2.

Immunoblot analysis of natural and vaccine-induced IgG responses to rubella virus proteins expressed in insect cells

BACKGROUNDnThe three structural proteins of rubella virus (RV), the capsid protein C and the envelope glycoproteins E1 and E2, were produced individually in soluble form in Sf9 insect cells using the baculovirus system. All proteins were equipped with a polyhistidine tag at their C-terminal ends to enable gentle purification by metal ion affinity chromatography. In addition, the E1 and E2 proteins were engineered to display the FLAG epitope tag at their N-terminal ends.nnnSTUDY DESIGNnThe diagnostic potential of the recombinant purified proteins was evaluated by immunoblot and enzyme immuno assays (EIA) using a total of 57 well-characterised serum samples obtained at various time points after natural RV infection, congenital rubella syndrome (CRS), MMR vaccination or from controls with past RV immunity. In addition, acute and convalescent phase serum pools from a total of 20 patients were evaluated. Authentic RV proteins were used as a reference.nnnRESULTSnThe recombinant E1 and C proteins were predominant in eliciting the immune response in both postnatal and vaccinal RV infections, being much weaker in the vaccinal ones. The IgG response to the recombinant C protein was very strong after the first month post infection and decreased with time. The immune response against the recombinant E2 protein, however, was generally poor, but notably stronger after congenital infection. Together, the results showed that the individual recombinant protein antigens could be suitable for diagnosis of RV infection and for study of the immune response to rubella vaccination.

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 LMP1u2009C 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.

ANALYSIS OF HUMAN TROPHOBLASTIC TISSUE IN DIAGNOSIS OF EMBRYONIC VIRAL INFECTIONS

Summary Thirty four pregnant women with serologically and by isolation (from cervix and urine) proven viral infection (20 with CMV, 7 with HSV tip II, 6 with VZV and one with Parvovirus B19 infection) were studied. Chorionic villus sampling and amniocentesis were performed between 9th and 13th gestational week. Isolation of viruses on the culture of human fibroblasts for CMV, and on monkeys kidney for HSV was performed. Testing for indirect immunofluorescence with anti VZV specific serum and FITC marked antihuman globulin was used for detection of VZV antigens. The presence of HSV antigens was investigated by direct immunofluorescence with monoclonal antibodies on HSV type I and II, with FITC marked antibodies. CMV antigens was detected by indirect immunofluorescence. ELISA testing was used for Parvovirus B19. Anti HSV antibodies were detected by indirect immunofluorescence. ELISA testing also was used for detection of specific anti CMV IgG and IgM antibodies. Embryonic infection was detected in one case of VZV and Parvovirus B19 infection, two cases of HSV infection and six pregnancies with CMV infection (17%, 100%, 29%, 30%, respectively). In two cases of proven embryonic infection (VZV and CMV), the pregnancy was terminated, and in one (Parvovirus) spontaneous abortion occurred. Other pregnancies were continued. Cordocentesis were performed after the 22nd gestational week. The presence of specific and nonspecific signs of fetal infection was evaluated. Fetal infection was found in two (8%-CMV and HSV) in which embryonic infection could not have been proven (false negative results). One false positive (CMV) finding was found. It may be concluded that trophoblast analysis is important for studying of infections produced by teratogenic agents during pregnancy. It contributes to the early diagnosis of such infections (sensitivity 81%). Excluding the presence of infection (specificity 96%) is of even greater importance. The predictive value of the positive finding (90%) suggests that the positive findings should greatly influence decisions involving the subsequent therapeutical protocol. Negative findings (predictive value 92%) mandate application of additional diagnostic procedures in order to rule out any fetal-related risk.

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

BACKGROUNDnThree 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.nnnOBJECTIVESnThis 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.nnnSTUDY DESIGNnThe IL28B genotypes at 3 SNPs were determined in 106 patients who underwent standard 48-week therapy and out of which 55.7% achieved SVR.nnnRESULTSnPatients 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.nnnCONCLUSIONSnResults 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.