Echovirus 30 in Bulgaria during the European Upsurge of the Virus, 2017-2018.

In 2018, an increase in echovirus 30 (E30) detections was reported in some European countries. To assess the circulation and phylogenetic relationships of E30 in Bulgaria, E30 samples identified at the National Reference Laboratory for Enteroviruses, National Centre of Infectious and Parasitic Diseases, Bulgaria (NRL for Enteroviruses) in 2017 and 2018 were subjected to sequencing and phylogenetic analysis. The present study revealed that sample positivity did not significantly increase in Bulgaria during the European upsurge. E30 was identified in six patients, two of whom were epidemiologically linked. The maximum-likelihood phylogenetic tree showed that sequences from five patients belonged to the G1 lineage (clades G1a and G1b). The sequence from one patient belonged to the G2 lineage and was grouped closer to sequences from the last E30 outbreak in Bulgaria in 2012. No recombination events were detected. The European E30 upsurge in 2018 was caused by two clades, and one of them was G1. The fact that the majority of the Bulgarian samples belonged to G1 indicated that the virus was present in the country but did not cause a local upsurge. Phylogenetic and epidemiological data indicated sporadic E30 cases and a possible shift towards G1 lineage in 2017 and 2018.

High vaccine confidence and strong approval of the mandatory immunization schedule among Bulgarian general practitioners in 2022.

In a context of recently decreasing childhood immunization coverage and low uptake of COVID-19 vaccines in Bulgaria, this study measures vaccine hesitancy among general practitioners (GPs) in the country, as they are central to forming patients' attitudes. In 2022, a face-to-face survey was conducted through a simple random sample from an exhaustive national database of Bulgarian GPs. This study measured attitudes on vaccine importance, safety, and effectiveness, and attitudes toward the Bulgarian immunization schedule. Information was collected on demographic and GP practice characteristics and possible predictors of vaccine confidence in order to test for associations with attitudes toward immunization. GP attitudes toward vaccines and the immunization schedule in Bulgaria were generally positive. Among 358 respondents, 351 (98%,95%CI96-99%) strongly agreed/agreed that vaccines are important, 352 (98%,95%CI96-99%) that vaccines are effective, and 341 (95%,95%CI93-97%) that vaccines are safe. 347 respondents (97%,95%CI95-98%) affirmed that "it's good that vaccines from the children's immunization schedule are mandatory", and 331 (92%,95%CI89-95%) agreed with the statement "Bulgaria's childhood immunization has my approval". Trust in information from official institutions was among the strongest predictors of vaccine confidence. Respondents' vaccine confidence levels are within the ranges reported by GPs in other European countries and above those reported within the general Bulgarian population. GPs' vaccine confidence is highly associated with trust in official institutions. It is important to maintain trust in official institutions and to support GPs in communicating vaccine knowledge with patients so that vaccine hesitancy in the general population is countered.

Efficacy and safety of Aviron Rapid® in adolescents and children with viral acute upper respiratory tract infection: a multi-center, randomized, double blind, placebo-controlled clinical trial.

INTRODUCTION: Acute upper respiratory tract infections (AURTIs) are associated with a significant burden on society attributed to medical care and loss of productivity. Novel therapies that are able to shorten disease duration, while providing symptom relief and being well tolerated, are an unmet medical need.

Bulgarian General Practitioners' Communication Styles about Child Vaccinations, Mainly Focused on Parental Decision Making in the Context of a Mandatory Immunization Schedule.

The communication practices of general practitioners in relation with vaccines have not been a topic of wide scientific interest. In this article, we outline them in the context of Bulgaria. A representative, cross-sectional, quantitative, face-to-face survey was conducted among 358 Bulgarian general practitioners in 2022 using simple random sampling. We conducted an exploratory factor analysis using questions about the role of the GPs, which measure models of communication. Based on the factor analysis, we distinguished four communication styles. They were called: active communicator, restrictive communicator, informing communicator, and strained communicator. One-way ANOVA and the T-test were carried out to explore the connections between factor scores (communication styles) and other variables. One of the most important results in the study was that the informing physician (emphasizing the choice of the parents) was the most common model in Bulgaria. This is somewhat contradictory, because of the mandatory status of most vaccines. We found connections between the communication styles and other variables-such as the type of settlement, having a hesitant parent in the practice, recommendations of non-mandatory vaccines, and experience with vaccine-preventable diseases. On the basis of the factor analysis and analysis of relationships with other variables, we reached the conclusion that in Bulgaria, hesitant parents are not sufficiently involved in active, effective communication about vaccines by GPs.

Rhinovirus Genotypes Circulating in Bulgaria, 2018-2021.

Rhinoviruses (RV) are one of the most common causative agents of respiratory infections, with significant socioeconomic impact. RV infections are not notifiable in Bulgaria, and little is known about the different RV genotypes circulating in the country. This study aims to investigate the diversity of RV genotypes that were circulating in Bulgaria in the period 2018-2021 in samples from ILI/ARI patients. Genotype assignment was based on sequencing and phylogenetic analysis of the 5' untranslated region and the VP4-VP2 region. Out of a total of 1385 nasopharyngeal swabs tested, 166 were RV-positive (RV detection rate: 11.99% (166/1385)). Those with a cycle threshold <25 were selected for genotyping (n = 63). RV isolates were successfully genotyped and classified into 34 genotypes within Rhinovirus A (RV-A), Rhinovirus B (RV-B) and Rhinovirus C (RV-C) species. Presumptive recombination events between the 5'UTR and VP4-VP2 regions were detected in three of the isolates. RV-A and RV-C were the prevalent RV species, with significantly more frequent detections of RV-A in the years before the COVID-19 pandemic compared to the post-pandemic period, when RV-C prevailed. The present study is the first to determine RV genotypes in Bulgaria and the circulation of RV-C has been described for the first time in the country.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Retrospective Analysis of Six Years of Acute Flaccid Paralysis Surveillance and Polio Vaccine Coverage Reported by Italy, Serbia, Bosnia and Herzegovina, Montenegro, Bulgaria, Kosovo, Albania, North Macedonia, Malta, and Greece.

Here we analyzed six years of acute flaccid paralysis (AFP) surveillance, from 2015 to 2020, of 10 countries linked to the WHO Regional Reference Laboratory, at the Istituto Superiore di Sanità, Italy. The analysis also comprises the polio vaccine coverage available (2015-2019) and enterovirus (EV) identification and typing data. Centralized Information System for Infectious Diseases and Laboratory Data Management System databases were used to obtain data on AFP indicators and laboratory performance and countries' vaccine coverage from 2015 to 2019. EV isolation, identification, and typing were performed by each country according to WHO protocols. Overall, a general AFP underreporting was observed. Non-Polio Enterovirus (NPEV) typing showed a high heterogeneity: over the years, several genotypes of coxsackievirus and echovirus have been identified. The polio vaccine coverage, for the data available, differs among countries. This evaluation allows for the collection, for the first time, of data from the countries of the Balkan area regarding AFP surveillance and polio vaccine coverage. The need, for some countries, to enhance the surveillance systems and to promote the polio vaccine uptake, in order to maintain the polio-free status, is evident.

Poliovirus Antibody Seroprevalence among Laboratory Staff at the National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria.

BACKGROUND: The world is on the verge of eradicating polio. In the absence of immunity, laboratory staff handling infectious clinical specimens or viable polioviruses may eventually become a source for transmission. Polio vaccine is mandatory in Bulgaria. Control of acquired immunity is carried out only sporadically. As antibody titers decline with age, determining the seroprevalence in adult laboratory staff would be a contribution to the risk assessment in case of polio importation. METHODS: Cell culture microneutralization assay for detecting antibodies against poliovirus 1 and 3 was applied. RESULTS: The seroprevalence of poliovirus 1 and 3 antibodies among personnel employed at the National Center of Infectious and Parasitic Diseases in Bulgaria, who are handling stool specimens, was 100 and 79%, respectively. CONCLUSIONS: Seroprevalence meets the target of 80%. It can be concluded that personnel are protected against polioviruses and would not be a source of infection in case of polio importation.

Preliminary findings indicate nosocomial transmission and Roma population as most affected group in ongoing measles B3 genotype outbreak in Bulgaria, March to August 2017.

From March to August 2017, 165 measles cases were reported from three regions in Bulgaria. The age range was 0-55 years and 66% of the cases were under 9 years. The Roma population was disproportionally affected (89% of cases), 41% cases were unvaccinated and in 24 cases there was nosocomial transmission mostly in paediatric departments. A child under 12 months of age died. Control measures have been taken and the investigation is still ongoing.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Serological and Molecular Biological Studies of Parvovirus B19, Coxsackie B Viruses, and Adenoviruses as Potential Cardiotropic Viruses in Bulgaria.

BACKGROUND: Inflammatory diseases of the heart (myocarditis, pericarditis) are commonly caused by viruses. Among the human cardiotropic viruses, parvovirus B19, Coxsackie B viruses, and adenoviruses play a leading role. AIM: The aim of the present study was to determine the presumptive causative role of parvovirus B19, Coxsackie B viruses, and adenoviruses in the development of myocarditis, pericarditis and dilated cardiomyopathy by demonstrating the presence of specific antiviral antibodies or viral DNA in patients' serum samples. MATERIALS AND METHODS: We tested serum samples collected between 2010 and 2014 from 235 patients with myocarditis (n=108), pericarditis (n=79), myopericarditis (n=19), dilated cardiomyopathy (n=7), and fever of unknown origin accompanied by cardiac complaints (n=22). The mean age of patients with the standard deviation was 33 ± 18 years. Serological and molecular methods (ELISA for specific IgM/IgG antibodies to parvovirus B19 and IgM antibodies to Coxsackie B viruses and adenoviruses, and PCR for detection of parvovirus B19 in serum samples, respectively) were used in the study. RESULTS: Of all tested 235 serum samples, in 60 (25.5%) positive results for at least one of the three tested viruses were detected. Forty out of these 235 serum samples (17%) were Coxsackie B virus IgM positive. They were found in 17% (18/108) of the patients with myocarditis, in 15% (12/79) of those with pericarditis, in 16% (3/19) of those with myopericarditis and in 32% (7/22) in those with fever of unknown origin. The 63 Coxsackie B virus IgM negative patient's serum samples were tested by ELISA for presence of adenovirus IgM antibodies. Such were found in 4 patients with pericarditis and in 2 patients with fever of unknown origin. Every IgM negative sample (n=189) for Coxsackie B and adenovirus was further tested by ELISA for parvovirus B19 IgM/IgG antibodies. B19-IgM antibodies were detected in 14 patients (7.4%). The percentages for B19-IgM antibodies was 8% (7/90), 5% (3/63) and 31% (4/13) in the patients affected with myocarditis, pericarditis, and fever of unknown origin, respectively. Protective B19-IgG antibodies were found in 108 (57%) of the samples. A B19-PCR signal was detected in all the patients who were B19-IgM positive, and in only 1 patient with positive B19-IgG result, the latter presenting with dilated cardiomyopathy. CONCLUSION: The present study shows the involvement of Coxsackie B, parvovirus B19 and adenoviruses in the development of inflammatory diseases of the heart (myocarditis and pericarditis). It is the first ever study in the country that simultaneously analyzes the prevalence of the three major human cardiotropic viruses.

In vitro antiviral activity of a series of wild berry fruit extracts against representatives of Picorna-, Orthomyxo- and Paramyxoviridae.

Wild berry species are known to exhibit a wide range of pharmacological activities. They have long been traditionally applied for their antiseptic, antimicrobial, cardioprotective and antioxidant properties. The aim of the present study is to reveal the potential for selective antiviral activity of total methanol extracts, as well as that of the anthocyanins and the non-anthocyanins from the following wild berries picked in Bulgaria: strawberry (Fragaria vesca L.) and raspberry (Rubus idaeus L.) of the Rosaceae plant family, and bilberry (Vaccinium myrtillis L.) and lingonberry (Vaccinium vitis-idaea L) of the Ericaceae. The antiviral effect has been tested against viruses that are important human pathogens and for which chemotherapy and/or chemoprophylaxis is indicated, namely poliovirus type 1 (PV-1) and coxsackievirus B1 (CV-B1) from the Picornaviridae virus family, human respiratory syncytial virus A2 (HRSV-A2) from the Paramyxoviridae and influenza virus A/H3N2 of Orthomyxoviridae. Wild berry fruits are freeze-dried and ground, then total methanol extracts are prepared. Further the extracts are fractioned by solid phase extraction and the non-anthocyanin and anthocyanin fractions are eluted. The in vitro antiviral effect is examined by the virus cytopathic effect (CPE) inhibition test. The results reveal that the total extracts of all tested berry fruits inhibit the replication of CV-B1 and influenza A virus. CV-B1 is inhibited to the highest degree by both bilberry and strawberry, as well as by lingonberry total extracts, and influenza A by bilberry and strawberry extracts. Anthocyanin fractions of all wild berries strongly inhibit the replication of influenza virus A/H3N2. Given the obtained results it is concluded that wild berry species are a valuable resource of antiviral substances and the present study should serve as a basis for further detailed research on the matter.

Chemical and antiviral study on alkaloids from Papaver pseudocanescens M. Pop.

The phytochemical investigation of the aerial parts of Papaver pseudocanescens M. Pop. of Mongolian origin resulted in the isolation and structural elucidation of 8 alkaloids of the isoquinoline and promorphinane type. 8,14-Dihydroamurine, 8,14-dihydroflavinantine, and flavinantine are promorphinanes. Alborine, mecambridine, and mecambridine methohydroxide are retroprotoberberines. Amurensinine is an isopavine alkaloid and O-methylarmepavine is a benzylisoquinoline alkaloid. O-Methylarmepavine is a new alkaloid for the genus Papaver. Promorphinane-type alkaloids have been found for the first time in the species. All structures were established by physical and spectral analysis. As a first attempt to describe some of the biological activities of these alkaloids, the antiviral effect was tested against the in vitro replication of several viruses which belong to different taxonomic groups and represent significant human pathogens. Based on the results, the conclusion could be drawn that particular alkaloids from P. pseudocanescens possess selective antiviral effects against the replication of poliovirus 1 and human rhinovirus 14, two viruses from the Enterovirus genus of the Picornaviridae family.

Identification of glycosylated sites in Rapana hemocyanin by mass spectrometry and gene sequence, and their antiviral effect.

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

Synergistic drug combinations against the in vitro replication of Coxsackie B1 virus.

The existence of synergistic drug combinations against the in vitro replication of poliovirus type 1 (Mahoney) (PV-1) had been established in our previous work. The objective of the present study was to test the combined effects of the different drugs against another representative of the enterovirus genus, i.e. Coxsackievirus B1 (CBV-1). Dual combinations of enviroxime, disoxaril, arildone, PTU-23, HBB and S-7 were evaluated. The susceptibility of CBV-1 to the individual effects of the inhibitors was compared to that of PV-1. CBV-1 was more sensitive to enviroxime, S-7, PTU-23 and HBB and less sensitive to the effects of disoxaril and arildone. The effect of most dual drug combinations tested against CBV-1 replication was additive or synergistic. Enviroxime and S-7, enviroxime and PTU-23, disoxaril and HBB, disoxaril and PTU-23, arildone and HBB, arildone and PTU-23, S-7 and HBB revealed a strong synergistic effect. Synergy against CBV-1 replication was stronger as compared to that noted for the same drug combinations against PV-1 replication.