No abstract

BackgroundHepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies.MethodsThe HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested.ResultsWe confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs.ConclusionsWe describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

Viral hepatitis, particularly hepatitis B and C, are diseases with worldwide distribution that present a significant public health problem. Seroprevalence studies allow assessment of the extent of the disease burden, the identification of populations at risk and the monitoring trends over time. A multi-center seroprevalence study, carried out in Bulgaria (covering the five largest cities - Sofia, Plovdiv, Varna, Pleven, and Stara Zagora) in 1999-2000 estimated a crude seroprevalence rate of 3.9% for HBsAg and 1.3% for anti-HCV. A decade later, comparable rates were observed in a study including 865 outpatients consulting a clinical laboratory in Plovdiv, the second largest administrative region in Bulgaria. The crude seroprevalence rate measured for hepatitis B (HBsAg) was 3.9%. The HBsAg prevalence rate in individuals ≤19 years of age (targeted by vaccination) was significantly lower compared to the rate in adults ≥20 years of age -1% versus 4.8%. The lack of dynamics in the overall level of HBsAg carriers is likely related to the excessively low hepatitis B vaccine coverage in individuals, born before the introduction of the universal vaccination of newborns in August 1991. Anti-HCV antibodies were detected in 0.7% of the subjects.

Intravenous drug users constitute a group at risk for hepatitis C virus (HCV) infection. Today, no data are available on the molecular epidemiology of HCV in Bulgaria despite the fact that in recent years the incidence of acute hepatitis C infection among Bulgarian intravenous drug users increased sixfold and about 2/3 of them developed a chronic infection. The aim of this study was to determine the circulation of hepatitis C genotypes among drug users and to study the evolution and transmission history of the virus by molecular clock and Bayesian methods, respectively. Sequencing of NS5B gene showed that the genotype 3a was the most prevalent type among intravenous drug users. In the Bayesian tree, the 3a subtypes grouped in one main clade with one small cluster well statistically supported. The root of the tree was dated back to the year 1836, and the main clade from Bulgaria was dated 1960. The effective number of infections remained constant until about years 1950s, growing exponentially from the 1960s to the 1990s, reaching a plateau in the years 2000. The not significant intermixing with isolates from other countries may suggest a segregated circulation of the epidemic between 1940s and 1980s. The plateau reached by the epidemic in the early 2000s may indicate the partial success of the new preventive policies adopted in Bulgaria. J. Med. Virol. 83:1565-1570, 2011. © 2011 Wiley-Liss, Inc

BackgroundHepatitis A virus (HAV) infection is endemic in Eastern European and Balkan region countries. In 2012, Bulgaria showed the highest rate (67.13 cases per 100,000) in Europe. Nevertheless, HAV genotypes and strains circulating in this country have never been described. The present study reports the molecular characterization of HAV from 105 patients from Bulgaria.MethodsAnti-HAV IgM positive serum samples collected in 2012–2014 from different towns and villages in Bulgaria were analysed by nested RT-PCR, sequencing of the VP1/2A region and phylogenetic analysis; the results were analysed together with patient and geographical data.ResultsPhylogenetic analysis revealed two main sequence groups corresponding to the IA (78/105, 74%) and IB (27/105, 26%) sub-genotypes.In the IA group, a major and a minor cluster were observed (62 and 16 sequences, respectively). Most sequences from the major cluster (44/62, 71%) belonged to either of two strains, termed "strain 1" and "strain 2", differing only for a single specific nucleotide; the remaining sequences (18/62, 29%) showed few (1 to 4) nucleotide variations respect to strain 1 and 2. Strain 2 is identical to the strain previously responsible for an outbreak in the Czech Republic in 2008 and a large multi-country European outbreak caused by contaminated mixed frozen berries in 2013.Most sequences of the IA minor cluster and the IB group were detected in large/medium centers (LMCs). Overall, sequences from the IA major cluster were more frequent in small centers (SCs), but strain 1 and strain 2 showed an opposite relative frequency in SCs and LMCs (strain 1 more frequent in SCs, strain 2 in LMCs).ConclusionsGenotype IA predominated in Bulgaria in 2012–2014 and phylogenetic analysis identified a major cluster of highly related or identical IA sequences, representing 59% of the analysed cases; these isolates were mostly detected in SCs, in which HAV shows higher endemicity than in LMCs. The distribution of viral sequences suggests the existence of some differences between the transmission routes in SCs and LMCs.Molecular characterization of an increased number of isolates from Bulgaria, regularly collected over time, will be useful to explore specific transmission routes and plan appropriate preventing measures.

The study demonstrates the presence of immune memory and protection 5-15 years after the initial course of newborn immunization with recombinant vaccines against hepatitis B.

Hepatitis B virus infection is a global health problem. Based on the sequence divergence of the entire genome, hepatitis B virus has been classified into eight genotypes which have a characteristic geographic distribution. To date, no data are available on the molecular epidemiology of hepatitis B virus in Bulgaria. The aim of the present study was to reconstruct the epidemiological history of HBV genotypes/subgenotypes circulating in Bulgaria using a phylodynamic approach and a Bayesian statistical inference framework. Sequence analysis of the HBsAg/Reverse Transcriptase overlapping genomic regions revealed that D1 and A2 were the subgenotypes detected most frequently in the patients examined. The tMRCA estimations of the few HBV D1 Bulgarian significant clades dated back to 23-27 years ago, corresponding to the early 1980s. The HBV A2 Bulgarian sequences fell into two closely related supported clusters dated to 2003 and 1996 years, respectively, suggesting a more recent introduction of subgenotype A2 into Bulgaria. The study provides new information about the HBV subgenotypes in Bulgaria.

In a representative nationwide study, we have determined the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfections among HIV-positive patients diagnosed during the period 2010-2014 in Bulgaria. Despite a relatively low rate of new HIV diagnoses, the rates of hepatitis B and C coinfections among these patients fell within the upper range reported in Europe. HBsAg and HCV antibodies (Ab) were found in 10.4% and 25.6% of the tested individuals, respectively. Importantly, high rates of active hepatitis infections were confirmed by detection of HBV DNA in 51.1% and HCV RNA in 78.1% of the tested individuals. Hepatitis coinfections affected mostly high risk groups and persons with multiple risk behavior, including people who inject drugs, men who have sex with men, prisoners, and Roma people.