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Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6â%) patients. Of these, 364 (52.4â%), 213 (30.7â%) and 117 (16.9â%) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.
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Variação Genética , Vírus da Influenza B , Influenza Humana , Neuraminidase , Filogenia , SARS-CoV-2 , Humanos , Influenza Humana/virologia , Influenza Humana/epidemiologia , Vírus da Influenza B/genética , Vírus da Influenza B/classificação , Vírus da Influenza B/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/classificação , Neuraminidase/genética , Adulto , Masculino , Pessoa de Meia-Idade , Feminino , Bulgária/epidemiologia , Adulto Jovem , Idoso , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Pré-Escolar , Criança , Adolescente , COVID-19/epidemiologia , COVID-19/virologia , Lactente , Estações do Ano , Vírus da Influenza A/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificaçãoRESUMO
This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.
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COVID-19 , Coinfecção , Filogenia , SARS-CoV-2 , Humanos , Coinfecção/virologia , Coinfecção/epidemiologia , SARS-CoV-2/genética , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , COVID-19/epidemiologia , Pessoa de Meia-Idade , Adulto , Feminino , Masculino , Adolescente , Pré-Escolar , Criança , Idoso , Adulto Jovem , Lactente , Infecções Respiratórias/virologia , Infecções Respiratórias/epidemiologia , Rhinovirus/genética , Rhinovirus/classificação , Rhinovirus/isolamento & purificação , Vírus da Influenza A/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Vírus Sincicial Respiratório Humano/classificação , Nasofaringe/virologia , Sequenciamento Completo do Genoma , China/epidemiologia , Estações do Ano , Idoso de 80 Anos ou mais , Genoma Viral , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza B/classificaçãoRESUMO
Background: Respiratory syncytial virus (RSV) is a common cause of upper and lower respiratory tract infections. This study aimed to explore the prevalence of respiratory syncytial virus (RSV) and other respiratory viruses in Bulgaria, characterize the genetic diversity of RSV strains, and perform amino acid sequence analyses of RSV surface and internal proteins. Methods: Clinical and epidemiological data and nasopharyngeal swabs were prospectively collected from patients with acute respiratory infections between October 2020 and May 2023. Real-time PCR for 13 respiratory viruses, whole-genome sequencing, phylogenetic, and amino acid analyses were performed. Results: This study included three epidemic seasons (2020-2021, 2021-2022, and 2022-2023) from week 40 of the previous year to week 20 of the following year. Of the 3,047 patients examined, 1,813 (59.5%) tested positive for at least one viral respiratory pathogen. RSV was the second most detected virus (10.9%) after SARS-CoV-2 (22%). Coinfections between RSV and other respiratory viruses were detected in 68 cases, including 14 with SARS-CoV-2. After two seasons of low circulation, RSV activity increased significantly during the 2022-2023 season. The detection rates of RSV were 3.2, 6.6, and 13.7% in the first, second, and third seasons, respectively. RSV was the most common virus found in children under 5 years old with bronchiolitis (40%) and pneumonia (24.5%). RSV-B drove the 2022-2023 epidemic. Phylogenetic analysis indicated that the sequenced RSV-B strains belonged to the GB5.0.5a and GB5.0.6a genotypes. Amino acid substitutions in the surface and internal proteins, including the F protein antigenic sites were identified compared to the BA prototype strain. Conclusion: This study revealed a strong resurgence of RSV in the autumn of 2022 after the lifting of anti-COVID-19 measures, the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, and relatively low genetic diversity in circulating RSV strains.
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The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought about significant challenges worldwide. In this study, we present a comprehensive analysis of the genomic epidemiology and lineage dynamics of SARS-CoV-2 in Bulgaria over a three-year period. Through extensive genomic sequencing and data analysis, we investigated the evolution of the virus, the emergence of variants of concern (VOCs), and their impact on the country's pandemic trajectory. We also assessed the relationship between viral diversity and COVID-19 morbidity and mortality in Bulgaria. Our findings shed light on the temporal and spatial distribution of SARS-CoV-2 lineages and provide crucial insights into the dynamics of the pandemic in the country. The interplay between international travel and viral transmission plays a significant role in the emergence and dissemination of different SARS-CoV-2 variants. The observed proportions of exportation to various continents provide insights into the potential pathways through which these lineages spread globally. Understanding the genomic epidemiology of SARS-CoV-2 in Bulgaria is essential for formulating targeted public health strategies, enhancing vaccination efforts, and effectively managing future outbreaks.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Pandemias , Bulgária/epidemiologia , COVID-19/epidemiologia , GenômicaRESUMO
The first recombinant SARS-CoV-2 variants were identified in 2022, causing public health concerns. The importance of recombinant variants has increased especially since the WHO designated the recombinant variant XBB and its lineages as subvariants that require monitoring on 20 November 2022. In this study, we provide the first insights into the new SARS-CoV-2 variant named XAN, a recombinant composed of Omicron sub-lineages BA.2 and BA.5. To our knowledge, this is the first report on the recombinant SARS-CoV-2 XAN variant identified in Bulgaria.
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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.
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COVID-19 , Infecções por Enterovirus , Infecções por Picornaviridae , Infecções Respiratórias , Humanos , Rhinovirus , Filogenia , Bulgária/epidemiologia , Pandemias , COVID-19/genética , Genótipo , Infecções por Enterovirus/genética , Regiões 5' não TraduzidasRESUMO
Social distancing, mask-wearing, and travel restrictions during the COVID-19 pandemic have significantly impacted the spread of influenza viruses. The objectives of this study were to analyze the pattern of influenza virus circulation with respect to that of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Bulgaria during the 2021-2022 season and to perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains. Influenza infection was confirmed using real-time reverse transcription polymerase chain reaction in 93 (4.2%) of the 2193 patients with acute respiratory illness tested wherein all detected viruses were subtyped as A(H3N2). SARS-CoV-2 was identified in 377 (24.3%) of the 1552 patients tested. Significant differences in the incidence of influenza viruses and SARS-CoV-2 were found between individual age groups, outpatients/inpatients, and in the seasonal distribution of cases. Two cases of coinfections were identified. In hospitalized patients, the Ct values of influenza viruses at admission were lower in adults aged ≥65 years (indicating higher viral load) than in children aged 0-14 years (p < 0.05). In SARS-CoV-2-positive inpatients, this association was not statistically significant. HA genes of all A(H3N2) viruses analyzed belonged to subclade 3C.2a1b.2a. The sequenced viruses carried 11 substitutions in HA and 5 in NA, in comparison to the vaccine virus A/Cambodia/e0826360/2020, including several substitutions in the HA antigenic sites B and C. This study revealed extensive changes in the typical epidemiology of influenza infection, including a dramatic reduction in the number of cases, diminished genetic diversity of circulating viruses, changes in age, and seasonal distribution of cases.
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COVID-19 , Vacinas contra Influenza , Influenza Humana , Adulto , Criança , Humanos , Vírus da Influenza A Subtipo H3N2/genética , SARS-CoV-2/genética , Estações do Ano , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Bulgária/epidemiologia , Filogenia , Prevalência , Pandemias , COVID-19/epidemiologia , RNA Viral/genética , Análise de Sequência de DNA , Hemaglutininas , Neuraminidase/genéticaRESUMO
The Coronavirus Disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) affects mainly older adults. Those with comorbidities are at a higher risk of severe disease and even death. The symptomatic infection rate of children is lower, manifestations are milder, and severe forms are scarce. We present here two children with severe COVID-19 and a respiratory syncytial virus, with the goal of emphasizing the possibility of coinfection with a severe course and a different result. The microbiological diagnosis was made using multiplex PCR. This assay not only provided an early and accurate diagnosis but also aided in the implementation of contact precautions. Further research should be done to determine the influence of coinfection on the clinical course and outcome of pediatric patients.
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COVID-19 , Coinfecção , Influenza Humana , Infecções por Vírus Respiratório Sincicial , Infecções Respiratórias , Humanos , Criança , Idoso , SARS-CoV-2 , COVID-19/complicações , Infecções Respiratórias/diagnóstico , Infecções por Vírus Respiratório Sincicial/complicações , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sinciciais RespiratóriosRESUMO
BACKGROUND: The present study assesses the immune response in children with viral-induced wheezing by examining the two factors-interferon-gamma (IFN-γ) and periostin in serum and nasopharyngeal aspirate (NPA). The aim was to find a pattern with the severity and frequency of wheezing episodes. METHODS: Sixty-nine infants (40 boys and 29 girls), with a mean age of 11.4±6 (2 - 23) months, hospitalized with a first or recurrent episode of bronchial obstruction were enrolled in this study. The serum and NPA concentrations of IFN-γ and periostin were assessed by ELISA methodology. Fifty of the children (72%) were followed for 2 years. RESULTS: We detected lower NPA IFN-γ production in boys, infants with atopic status, family history of asthma, and respiratory syncytial virus infection. Recurrent wheezing in children was associated with a twice lower concentration of IFN-γ in NPA compared to those with the first episode (7.1 vs. 14.8 pg/ml, p=0.05). Higher serum periostin level was established in children over 12 mo in the group of recurrent wheezers with persistent manifestations compared to those without symptoms during the follow-up (410.5 vs. 269.7 ng/ml, p = 0.03). Multivariate logistical regression model assessed high level of serum periostin, male gender, atopy, family history of asthma, and severity of the attack as significant risk factors for persistent compared to intermittent wheezing (r < sup > 2 < /sup > = 0.87, p = 0.04). CONCLUSIONS: Our results demonstrated that recurrent viral-induced wheezing is associated with decreased IFN-γ production and increased periostin response and their correlation with severity and persistence of symptoms were the main outcome measures.
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Asma , Infecções por Vírus Respiratório Sincicial , Criança , Feminino , Lactente , Humanos , Masculino , Pré-Escolar , Adolescente , Interferon gama , Sons Respiratórios/etiologia , Seguimentos , Infecções por Vírus Respiratório Sincicial/complicações , Asma/complicaçõesRESUMO
Introduction: This study aimed to determine the prevalence, viral profile, and clinical features of coinfections with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and other respiratory viruses. Methods: Nasopharyngeal samples and clinical data of 221 hospitalized patients and 21 outpatients were collected and analyzed. Real-time reverse transcription-polymerase chain reaction was used to detect SARS-CoV-2, influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus (PIV) 1,2,3, rhinovirus (RV), adenovirus (AdV), bocaviruses (BoV), and seasonal coronaviruses (OC43, 229E, NL63, and HKU1). Viral load was determined by capillary electrophoresis. Results: From November 2020 to mid-March 2022, 242 SARS-CoV-2 positive patients were tested for seasonal respiratory viruses, and 24 (9.9%) cases of coinfections were detected. The distribution of viruses involved in cases of coinfections were as follows: HMPV (n = 6; 25%), RSV (n = 4;16.7%), AdV (n = 4; 16.7%), BoV (n = 4; 16.7%), PIV3 (n = 2; 8.3%), influenza A (H3N2; n = 2; 8.3%), RV (n = 1; 4.62%), and RV+BoV (n = 1; 4.62%). The proportion of detected coinfections with SARS-CoV-2 was highest in children aged 0-5 years (59%), followed by those >65 years (33%). In specimens with detected coinfection, the viral load of influenza was higher than that of SARS-CoV-2, and the mean viral load of SARS-CoV-2 was higher than that of the other respiratory viruses. C-reactive protein (CRP) and lymphocytes count in co-infected patients >65 years of age were on average higher than in children <16 years of age (mean CRP of 161.8 ± 133.1 mg/L; 19.7 ± 3.09% vs. mean 6.9 ± 8.9 mg/L, 0.9 ± 3.1%; p < 0.01). Patients >65 years of age co-infected with SARS-CoV-2 and other respiratory viruses had longer hospital stays than those <16 years of age (mean 9 ± 3.96 days vs. 5.44 ± 1.89 days; p = 0.025). The combination of AdV and SARS-CoV-2 is fatal for patients aged >65 years. Conclusion: In patients aged >65 years, coinfection with SARS CoV-2 and other respiratory viruses, together with concomitant diseases, causes worsening of the clinical picture and complications, and can be fatal. Screening of patients with SARS CoV-2 for other respiratory viruses is needed to select appropriate treatments and prevent a fatal outcome of the disease.
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COVID-19 , Coinfecção , Influenza Humana , Adolescente , Idoso , Proteína C-Reativa , COVID-19/epidemiologia , Criança , Coinfecção/epidemiologia , Humanos , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/epidemiologia , SARS-CoV-2RESUMO
Ðuman bocaviruses (hBoVs) are often associated with acute respiratory infections (ARIs). Information on the distribution and molecular epidemiology of hBoVs in Bulgaria is currently limited. The objectives of this study were to investigate the prevalence and genetic characteristics of hBoVs detected in patients with ARIs in Bulgaria. From October 2016 to September 2019, nasopharyngeal/oropharyngeal swabs were prospectively collected from 1842 patients of all ages and tested for 12 common respiratory viruses using a real-time RT-PCR. Phylogenetic and amino acid analyses of the hBoV VP1/VP2 gene/protein were performed. HBoV was identified in 98 (5.3%) patients and was the 6th most prevalent virus after respiratory-syncytial virus (20.4%), influenza A(H1N1)pdm09 (11.1%), A(H3N2) (10.5%), rhinoviruses (9.9%), and adenoviruses (6.8%). Coinfections with other respiratory viruses were detected in 51% of the hBoV-positive patients. Significant differences in the prevalence of hBoVs were found during the different study periods and in patients of different age groups. The detection rate of hBoV was the highest in patients aged 0-4 years (6.9%). In this age group, hBoV was the only identified virus in 9.7%, 5.8%, and 1.1% of the children diagnosed with laryngotracheitis, bronchiolitis, and pneumonia, respectively. Among patients aged ≥5 years, hBoV was detected as a single agent in 2.2% of cases of pneumonia. Phylogenetic analysis showed that all Bulgarian hBoV strains belonged to the hBoV1 genotype. A few amino acid substitutions were identified compared to the St1 prototype strain. This first study amongst an all-age population in Bulgaria showed a significant rate of hBoV detection in some serious respiratory illnesses in early childhood, year-to-year changes in the hBoV prevalence, and low genetic variability in the circulating strains.
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INTRODUCTION: We investigated the prevalence of human metapneumovirus (hMPV) among patients with acute respiratory infections in Bulgaria, and performed genetic characterization of the F gene of these strains. METHODS: Nasopharyngeal swabs collected from patients of a range of ages were tested by using real-time PCR for 12 respiratory viruses. The F gene was sequenced, and phylogenetic and amino acid analyses of the F gene/protein were performed. RESULTS: A total of 1,842 patients were examined during a 3-year period; 1,229 patients (66.7%) were positive for at least one respiratory virus. hMPV was identified in 83 (4.5%) patient samples. Eleven (13%) of hMPV-positive patients were coinfected with another respiratory virus. The hMPV incidence rate in the 2016/2017, 2017/2018, and 2018/2019 winter seasons was 5.4, 5.4, and 3.1%, respectively. hMPV was mainly detected in specimens collected between January and May (89.2% of cases). The incidence of hMPV infection was highest (5.1%) among the youngest age-group (0-4 years), where hMPV was a causative agent in 8.1 and 4.8% of bronchiolitis and pneumonia cases, respectively. Among the patients aged ≥5 years, hMPV was detected in 2.2 and 3.2% of cases of pneumonia and central nervous system infections, respectively. Phylogenetic analysis of the F gene showed that the sequenced hMPV strains belonged to the A2b, B1, and B2 genotypes. Numerous amino acid substitutions were identified compared with the NL00/1 prototype strain. CONCLUSION: This study revealed the significant role of hMPV as a causative agent of serious respiratory illnesses in early childhood, and also demonstrated year-to-year changes in hMPV prevalence and genetic diversity in circulating strains.
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Metapneumovirus , Infecções por Paramyxoviridae , Infecções Respiratórias , Bulgária/epidemiologia , Pré-Escolar , Genótipo , Humanos , Lactente , Recém-Nascido , Metapneumovirus/genética , Infecções por Paramyxoviridae/epidemiologia , Filogenia , Prevalência , Infecções Respiratórias/epidemiologiaRESUMO
Influenza viruses have a high potential for genetic changes. The objectives of this study were to analyse influenza virus circulation in Bulgaria during the 2019/2020 season, to perform a phylogenetic and molecular analyses of the haemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains, and to identify amino acid substitutions compared to the current vaccine strains. Seasonal influenza viruses A(H3N2), A(H1N1)pdm09 and B/Victoria-lineage were detected using a real-time RT-PCR in 323 (23.3%), 149 (10.7%) and 138 (9.9%) out of 1387 patient samples studied, respectively. The HA genes of A(H3N2) viruses analysed belonged to clades 3C.3a (21 strains) and 3C.2a (5 strains): subclades 3C.2a1b + T131K, 3C.2a1b + T135K-B and 3C.2a1b + T135K-A. The clade 3C.3a and subclade 3C.2a1b viruses carried 5 and 14-17 substitutions in HA, as well as 3 and 9 substitutions in NA, respectively, in comparison with the A/Kansas/14/2017 vaccine virus, including some substitutions in the HA antigenic sites A, B, C and E. All 21 A(H1N1)pdm09 viruses sequenced fell into 6B.1A5A subclade. Amino acid sequence analysis revealed the presence of 7-11 substitutions in HA, compared to the A/Brisbane/02/2018 vaccine virus, three of which occurred in antigenic site Sb, along with 6-9 changes at positions in NA. All 10 B/Victoria-lineage viruses sequenced belonged to clade 1A with a triple deletion in HA1 (genetic group 1A(Δ3)B) and carried 7 and 3 substitutions in HA and NA, respectively, with respect to the B/Colorado/06/2017 vaccine virus. The results of this study confirm the rapid evolution of influenza viruses and the need for continuous antigenic and genetic surveillance.
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Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Influenza Humana/genética , Neuraminidase/genética , Orthomyxoviridae/genética , Substituição de Aminoácidos/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vacinas contra Influenza/genética , Vacinas contra Influenza/uso terapêutico , Influenza Humana/virologia , Orthomyxoviridae/classificação , Orthomyxoviridae/patogenicidade , Filogenia , Estações do AnoRESUMO
The objectives of this study were to investigate the prevalence of respiratory syncytial virus (RSV) infections in Bulgaria, to characterize the genetic diversity of the RSV strains, and to perform amino acid sequence analysis of the RSV G protein. Clinical, epidemiological data and nasopharyngeal swabs were prospectively collected from children aged less than 5 years presenting with acute respiratory infections from October 2016 to September 2018. Real-time polymerase chain reaction for 12 respiratory viruses, and sequencing, phylogenetic, and amino acid analyses of the RSV G gene/protein were performed. Of the 875 children examined, 645 (73.7%) were positive for at least one viral respiratory pathogen. RSV was the most commonly detected virus (26.2%), followed by rhinoviruses (15%), influenza A (H3N2) (9.7%), adenoviruses (9%), bocaviruses (7.2%), human metapneumovirus (6.1%), parainfluenza viruses 1/2/3 (5.8%), influenza type B (5.5%), and A(H1N1)pdm09 (3.4%). The detection rate for RSV varied across two winter seasons (36.7% vs 20.3%). RSV-B cases outnumbered those of the RSV-A throughout the study period. RSV was the most common virus detected in patients with bronchiolitis (45.1%) and pneumonia (24%). Phylogenetic analysis indicated that all the sequenced RSV-A strains belonged to the ON1 genotype and the RSV-B strains were classified as BA9 genotype. Amino acid substitutions at 15 and 22 positions of the HVR-2 were identified compared with the ON1 and BA prototype strains, respectively. This study revealed the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, the dominance of RSV-B, and relatively low genetic diversity in the circulating RSV strains.
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Genótipo , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Bulgária/epidemiologia , Pré-Escolar , Feminino , Variação Genética , Humanos , Lactente , Recém-Nascido , Masculino , Técnicas de Diagnóstico Molecular , Filogenia , Prevalência , Estudos Prospectivos , Infecções por Vírus Respiratório Sincicial/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Estações do Ano , Análise de Sequência de DNA , Proteínas Virais/genética , Viroses/classificação , Viroses/epidemiologiaRESUMO
Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines.Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness.Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented.Results. A(H1N1)pdm09 (66â%) predominated over A(H3N2) (34â%) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50â% falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six-ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four-nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences.Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.
Assuntos
Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Influenza Humana/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Antígenos Virais/genética , Bulgária/epidemiologia , Monitoramento Epidemiológico , Evolução Molecular , Variação Genética/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas , História do Século XXI , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/imunologia , Influenza Humana/história , Influenza Humana/virologia , Neuraminidase/genética , Filogenia , RNA Viral/genética , Estações do Ano , Análise de Sequência de DNA/métodosRESUMO
Acute lower respiratory infections (ALRIs) are a leading cause of morbidity and hospital admissions in children. This study aimed to determine the viral etiology of these infections in children aged < 5 years during three successive epidemic seasons in Bulgaria. Nasopharyngeal and throat specimens were collected from children with bronchiolitis and pneumonia during the 2015/2016, 2016/2017, and 2017/2018 seasons. The viral etiology was determined by individual real-time PCR assays against 11 respiratory viruses. Of the 515 children examined, 402 (78.1%) were positive for at least one virus. Co-infections with two and three viruses were found in 64 (15.9%) of the infected children. Respiratory syncytial virus (RSV) was the predominant pathogen (37.5%), followed by rhinoviruses (13.8%), metapneumovirus (9.1%), adenoviruses (7%), bocaviruses (7%), influenza A(H1N1)pdm09 (4.9%), A(H3N2) (4.3%), type B (4.1%), and parainfluenza viruses 1/2/3 (2.9%). RSV-B were more prevalent than RSV-A during the three seasons. At least one respiratory virus was identified in 82.6% and 70.1% of the children with bronchiolitis and pneumonia, respectively. Respiratory viruses, especially RSV, are principal pathogens of ALRIs in children aged < 5 years. Diagnostic testing for respiratory viruses using molecular methods may lead to the reduced use of antibiotics and may assist in measures to control infection.
Assuntos
Infecções Respiratórias/virologia , Viroses/virologia , Vírus/isolamento & purificação , Doença Aguda/terapia , Bulgária , Pré-Escolar , Feminino , Hospitalização , Humanos , Lactente , Masculino , Infecções Respiratórias/terapia , Estações do Ano , Viroses/terapia , Vírus/classificação , Vírus/genéticaRESUMO
PURPOSE: Influenza viruses are characterised by high variability, which makes them able to cause annual epidemics. The aim of this study is to determine the antigenic and genetic characteristics of influenza viruses circulating in Bulgaria during the 2016/2017 season. METHODOLOGY: The detection and typing/subtyping of influenza viruses were performed using real time RT-PCR. Results of antigenic characterisation, phylogenetic and amino acid sequence analyses of representative influenza strains are presented herein. RESULTS: The 2016/2017 season was characterised by an early start, an exclusive dominance of A(H3N2) viruses accounting for 93â% of total influenza virus detections, and a low circulation of A(H1N1)pdm09 (4.2â%) and type B (2.5â%) viruses. The analysed A(H3N2) viruses belonged to subclades 3C.2a (52â%) and 3C.2a1 (48â%); all studied A(H1N1)pdm09 and B/Victoria-lineage viruses belonged to subclades 6B.1 and 1A, respectively. The amino acid sequence analysis of 56 A(H3N2) isolates revealed the presence of substitutions in 18 positions in haemagglutinin (HA) as compared to the A/Hong Kong/4801/2014 vaccine virus, seven of which occurred in four antigenic sites, together with changes in 23 positions in neuraminidase (NA), and a number of substitutions in internal proteins PB2, PB1, PB1-F2, PA, NP and NS1. Despite the many amino acid substitutions, A(H3N2) viruses remained antigenically similar to the vaccine strain. Substitutions in HA and NA sequences of A(H1N1)pdm09 and B/Victoria-lineage strains were also identified, including in antigenic sites. CONCLUSION: The results of this study confirm the genetic variability of circulating influenza viruses, particularly A(H3N2), and the need for continued antigenic and molecular surveillance.
Assuntos
Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Adolescente , Adulto , Substituição de Aminoácidos , Bulgária/epidemiologia , Criança , Pré-Escolar , Monitoramento Epidemiológico , Evolução Molecular , Feminino , Variação Genética , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza B/classificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Masculino , Neuraminidase/genética , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Análise de Sequência de DNA , Adulto JovemRESUMO
The respiratory syncytial virus (RSV) is a leading cause of acute respiratory illnesses (ARI) in infants and young children. The objectives of this study were to investigate the RSV circulation among children aged <5 years in Bulgaria, to identify the RSV-A and RSV-B genotypes and to perform an amino acid sequence analysis of second hypervariable region (HVR2) of the G gene. During the 2014/15 and 2015/16 winter seasons, nasopharyngeal specimens of 610 children aged <5 years with ARI were tested using Real Time RT-PCR for influenza viruses, RSV, metapneumovirus, parainfluenza viruses, rhinoviruses and adenoviruses. Viral respiratory pathogens were detected in 429 (70%) out of 610 patients examined and RSV was the most frequently identified virus (26%) followed by influenza A(H1N1)pdm09 virus (14%) (p < .05). RSV was the most prevalent pathogen in patients with bronchiolitis (48%) and pneumonia (38%). In the 2014/15 season, RSV-A dominated slightly (53%), while in the next season RSV-B viruses prevailed more strongly (66%). The phylogenetic analysis based on the G gene indicated that all 21 studied RSV-A strains belonged to the ON1 genotype; the vast majority (96%) of the RSV-B strains were classified into BA9 genotype and only one - into BA10 genotype. All Bulgarian RSV-A and RSV-B sequences contained a 72-nt and a 60-nt duplication in the HVR2, respectively. The study showed the leading role of this pathogen as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, a shift from RSV-A to RSV-B subgroup dominance and relatively low genetic divergence in the circulating strains.
Assuntos
Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Bulgária/epidemiologia , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Masculino , Filogenia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vírus Sincicial Respiratório Humano/classificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estações do AnoRESUMO
Influenza virological surveillance is an essential tool for early detection of novel genetic variants of epidemiologic and clinical significance. The aim of this study was to determine the antigenic and molecular characteristics of influenza viruses circulating in Bulgaria during the 2015/2016 season. The season was characterized by dominant circulation of A(H1N1)pdm09 viruses, accounting for 66% of detected influenza viruses, followed by B/Victoria-lineage viruses (24%) and A(H3N2) viruses (10%). All sequenced influenza A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses belonged to the 6B.1, 3C.2a and 1A genetic groups, respectively. Amino acid analysis of 57 A(H1N1)pdm09 isolates revealed the presence of 16 changes in hemagglutinin (HA) compared to the vaccine virus, five of which occurred in four antigenic sites, together with 16 changes in neuraminidase (NA) and a number of substitutions in proteins MP, NP, NS and PB2. Despite the many amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically closely related to A/California/7/2009 vaccine virus. Bulgarian A(H3N2) strains (subclade 3C.2a) showed changes at 11 HA positions four of which were located in antigenic sites A and B, together with 6 positions in NA, compared to the subclade 3C.3a vaccine virus. They contained unique HA1 substitutions N171K, S312R and HA2 substitutions I77V and G155E compared to Bulgarian 3C.2a viruses of the previous season. All 20 B/Victoria-lineage viruses sequenced harboured two substitutions in the antigenic 120-loop region of HA, and 5 changes in NA, compared to the B/Brisbane/60/2008 vaccine virus. The results of this study reaffirm the continuous genetic variability of circulating seasonal influenza viruses and the need for continued systematic antigenic and molecular surveillance.
Assuntos
Antígenos Virais/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Neuraminidase/genética , Filogenia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos , Bulgária/epidemiologia , Criança , Pré-Escolar , Monitoramento Epidemiológico , Feminino , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H3N2/classificação , Influenza Humana/transmissão , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Estações do Ano , Análise de Sequência de DNARESUMO
AIM: Influenza virological surveillance is an essential tool for studying the evolution of influenza viruses as well as for annual updating of the vaccine composition. The aim of the present study is to analyse the circulation of the influenza viruses in Bulgaria during the four recent post-pandemic seasons. METHODS: A total of 3,681 respiratory samples from patients with influenza like illness (ILI) or acute respiratory illness (ARI) were tested for influenza viruses using Real Time RT-PCR. RESULTS: Influenza viruses were detected in 1,367 (37%) samples. Of those viruses, 941 (69%) were of type A and 426 (31%) of type B. Among the subtyped A viruses, 543 (60%) were A(H1N1)pdm09 and 369 (40%) A(H3N2). Co-circulation of all seasonal influenza types/subtypes was registered during each season, with the exception of A(H1N1)pdm09 virus in the 2011/12 season. In this study, data gathered from the antigenic and genetic analyses of influenza viruses, their antiviral susceptibility, and the epidemiological and clinical characteristics of the infections are presented. CONCLUSIONS: Yearly variations in the distribution and frequency of influenza types/subtypes and an annual shift of the predominant type/subtype were observed. In the seasons with predominant spread of A(H1N1)pdm09 virus - 2010/11 and 2013/14, a greater number of influenza-related pneumonia cases, ICU admissions and fatal cases was registered (p<0.05). The results of the present study confirm the need for continuous and comprehensive influenza surveillance.