Recommendations for respiratory syncytial virus surveillance at the national level.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infections and hospitalisations among young children and is globally responsible for many deaths in young children, especially in infants aged <6 months. Furthermore, RSV is a common cause of severe respiratory disease and hospitalisation among older adults. The development of new candidate vaccines and monoclonal antibodies highlights the need for reliable surveillance of RSV. In the European Union (EU), no up-to-date general recommendations on RSV surveillance are currently available. Based on outcomes of a workshop with 29 European experts in the field of RSV virology, epidemiology and public health, we provide recommendations for developing a feasible and sustainable national surveillance strategy for RSV that will enable harmonisation and data comparison at the European level. We discuss three surveillance components: active sentinel community surveillance, active sentinel hospital surveillance and passive laboratory surveillance, using the EU acute respiratory infection and World Health Organization (WHO) extended severe acute respiratory infection case definitions. Furthermore, we recommend the use of quantitative reverse transcriptase PCR-based assays as the standard detection method for RSV and virus genetic characterisation, if possible, to monitor genetic evolution. These guidelines provide a basis for good quality, feasible and affordable surveillance of RSV. Harmonisation of surveillance standards at the European and global level will contribute to the wider availability of national level RSV surveillance data for regional and global analysis, and for estimation of RSV burden and the impact of future immunisation programmes.

Circulation of influenza A and B in the Czech Republic from 2000-2001 to 2015-2016.

BACKGROUND: To improve national influenza vaccination recommendations, additional data on influenza A and B virus circulation are needed. Here, we describe the circulation of influenza A and B in the Czech Republic during 16 seasons. METHODS: This was a retrospective analysis of data collected from the 2000-2001 to 2015-2016 influenza seasons by the Czech Republic national influenza surveillance network. Influenza was confirmed and viral isolates subtyped by virological assays followed by antigen detection or by reverse transcriptase-polymerase chain reaction. RESULTS: Of 16,940 samples collected, 5144 (30.4%) were influenza-positive. Influenza A represented 78.6% of positive cases overall and accounted for more than 55.0% of all influenza cases in every season, except for 2005-2006 (6.0%). Both A/H1N1 and A/H3N2 were detected in most seasons, except for 2001-2002 and 2003-2004 (only A/H3N2), and 2007-2008 and 2009-2010 (only A/H1N1). Influenza B represented 21.4% of positive cases overall (range, 0.0-94.0% per season). Both influenza B lineages were detected in three seasons, a single B lineage in 11, and no B strain in two. For the 11 seasons where influenza B accounted for ≥20% of positive cases, the dominant lineage was Yamagata in six and Victoria in four. In the remaining season, the two lineages co-circulated. For two seasons (2005-2006 and 2007-2008), the B lineage in the trivalent influenza vaccine did not match the dominant circulating B lineage. CONCLUSIONS: In the Czech Republic, during the 2000-2001 to 2015-2016 influenza seasons, influenza virus circulation varied considerably. Although influenza A accounted for the most cases in almost all seasons, influenza B made a substantial, sometimes dominant, contribution to influenza disease.

Mumps in the Czech Republic in 2013: Clinical Characteristics, Mumps Virus Genotyping, and Epidemiological Links.

AIM: The aim of the study was to map the incidence of mumps in the Czech Republic in terms of clinical symptoms, epidemiological links, and characteristics of circulating genotypes. METHODS: Patients with suspected mumps examined in the Infectious Diseases Clinic of the Na Bulovce Hospital in 2013 were enrolled in the study. Buccal swab specimens were tested by means of nucleic acid detection (RT-qPCR) and when positive, they were cultured in tissue culture. Sequencing was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit and Genetic Analyzer 3500. The SeqScape software was used for the analysis of sequencing data and filtering out low quality reads. The phylogenetic analysis and genotyping were performed using the Mega 6 software. To generate the phylogenetic tree, all sequences were aligned by the MAFFT tool and the alignment obtained was edited using the BioEdit software. In all patients, selected biochemical markers (C-reactive protein, white blood cell count and serum amylase) were measured. The EPIDAT system used for reporting infectious diseases, record keeping, and data analysis in the Czech Republic was the source of statistical data. RESULTS: Eighty-nine patients with suspected mumps were examined in the Na Bulovce Hospital and 65 of them were laboratory confirmed with mumps: 40 males (61.5%) and 25 females (38.5%). The mean age of the study cohort was 25.9 years (median age of 23 years, age range from 10 to 73 years) and 14 patients were under 18 years of age. Thirty-four (52.3%) patients were vaccinated in childhood, 28 (43.1%) were unvaccinated, and for three persons, vaccination data were not available. A severe course of the disease was reported in 15 (23.1%) patients. Fourteen of them needed hospitalization because of orchitis (9 males) and meningitis (5 patients). One patient with orchitis was treated on an outpatient basis. The need for hospitalization tended to be lower in the unvaccinated patients (14.7% vs. 35.7%, p=0.076). In 2013, 1,553 cases of mumps were reported to the EPIDAT system. Of these, 640 were laboratory confirmed. The most often reported complications were orchitis (90 cases, i.e. 10.3%) and meningitis (21 cases, i.e. 1.4%). Orchitis was diagnosed in 30.3% of the unvaccinated and in 6.4% of the vaccinated males. Meningitis occurred in 3.1% of the unvaccinated and in 1.0% of the vaccinated patients. CONCLUSION: Despite the emergence of mumps among the vaccinated population, the present study has confirmed a positive effect of the vaccine, particularly on the incidence of complications and inflammatory markers. All 30 sequenced mumps virus strains were assigned to group G. A secondary vaccine failure due to waning immunity seems to be a plausible explanation for the rise in mumps cases.

[Phylogenetic analysis and genotyping of A/H3N2 Influenza viruses isolated from patients hospitalised with influenza-like illness symptoms in the na bulovce hospital in the season 2011/2012]. / Fylogenetická analýza a genotypizace viru chripky A/H3N2 izolovaných od pacientu hospitalizovaných s príznaky ILI (Influenza-Like Illnesses) v Nemocnici Na Bulovce v sezone 2011/2012.

Influenza A virus is an important cause of acute respiratory infections (ARI). Clinical manifestations of ARI vary from mild or moderate to life-threatening conditions requiring intensive care. Given the segmented genome, a large natural reservoir of other influenza virus subtypes, and antibody selection pressure in the population, the virus is variable and genetically unstable. The phylogenetic analysis and genotyping of A/H3N2 influenza viruses isolated from patients hospitalised with influenza-like illness symptoms in the Na Bulovce Hospital in the season 2011/2012 support the assumption that the pathogenicity is a polygenic trait modifiable by the host health status and seems not to be unambiguously associated with any specific mutations.

[Microneutralization assay in the diagnosis of influenza infection]. / Mikroneutralizace v diagnostice chripkové infekce.

Serology plays an important role in the diagnosis of influenza, particularly in the detection of post-vaccination and post-infection antibodies. When considering the range of diagnostic options, the serological method should be selected depending on the circumstances - whether single or paired serum samples are tested, whether adequate patient medical history data are available, whether epidemiological links are suspected, and, in particular, to what purpose the result will be used (differential diagnosis, post-infection follow-up, post-vaccination monitoring, etc.). The virus neutralization assay is one of the most sensitive and most objective serological tests, but it is highly dependent on the reaction balance and quality of the virus used. Determining the protective titer is crucial for the routine practice. Based on our experiments, we concluded that the virus neutralizing antibody titers are up to eight times as high in comparison with the hemaglutination inhibition test (HIT) or complement fixation reaction (CFR), but the correlation varies and is significantly influenced by interindividual variation in anti-neuraminidase antibodies and those against some internal proteins of influenza virus. We assume that the protective titer in the virus neutralization assay will be not less than 1:80. The predictive value of the titers below 1:40 is questionable.

Development and evaluation of a one-step real-time RT-PCR assay for universal detection of influenza A viruses from avian and mammal species.

The objective of our study was to develop and evaluate a TaqMan real-time RT-PCR (RRT-PCR) assay for universal detection of influenza A (IA) viruses. The primers and LNA-modified octanucleotide probe were selected to correspond to extremely conserved regions of the membrane protein (MP) segment identified by a comprehensive bioinformatics analysis including 10,405 IA viruses MP sequences, i.e., all of the sequences of the Influenza Virus Sequence database collected as of August 20, 2009. The RRT-PCR has a detection limit of approximately five copies of target RNA/reaction and excellent reaction parameters tested in four IA viruses reference laboratories. The inclusivity of the assay was estimated at both the bioinformatic and the experimental level. Our results predicted that this RRT-PCR assay was able to detect 99.5% of known human IA virus strains, 99.84% of pandemic influenza A (H1N1) strains, 99.75% of avian strains, 98.89% of swine strains, 98.15% of equine strains, and 100% of influenza A viruses of other origin.

[Basic epidemiological characteristics of influenza infection]. / Základní epidemiologické charakteristiky chripkové infekce.

Influenza is a relatively serious infection that affects hundreds of thousands of people every year in the Czech Republic alone, with unnecessary deaths as a possible outcome. Vaccination against influenza is the most important preventive measure. The highest incidence of seasonal influenza is reported in school-age children and young adults while the highest mortality is observed among the elderly. Pandemic influenza may significantly differ from seasonal influenza in these two aspects. Other epidemiological characteristics of seasonal influenza are presented and compared with those of 2009 pandemic A/H1N1 ("Mexican") influenza that caused the first pandemic of the 21st century.

Challenge of conducting a placebo-controlled randomized efficacy study for influenza vaccine in a season with low attack rate and a mismatched vaccine B strain: a concrete example.

BACKGROUND: Our aim was to determine the efficacy of a trivalent inactivated split virus influenza vaccine (TIV) against culture-confirmed influenza A and/or B in adults 18 to 64 years of age during the 2005/2006 season in the Czech Republic. METHODS: 6203 subjects were randomized to receive TIV (N = 4137) or placebo (N = 2066). The sample size was based on an assumed attack rate of 4% which provided 90% power to reject the hypothesis that vaccine efficacy (VE) was > or = 45%. Cases of influenza like illness (defined as fever (oral temperature > or =37.8 degrees C) plus cough and/or sore throat) were identified both by active (biweekly phone contact) and passive (self reporting) surveillance and nasal and throat swabs were collected from subjects for viral culture. RESULTS: TIV was well tolerated and induced a good immune response. The 2005/2006 influenza season was exceptionally mild in the study area, as it was throughout Europe, and only 46 culture-confirmed cases were found in the study cohort (10 influenza A and 36 influenza B). Furthermore among the B isolates, 35 were identified as B/Hong Kong 330/2001-like (B/Victoria/2/87 lineage) which is antigenically unrelated to the vaccine B strain (B/Yamagata/16/88 lineage). The attack rate in the vaccine group (0.7%) was not statistically significantly different from the attack rate in the placebo group (0.9%). CONCLUSION: Due to the atypical nature of the influenza season during this study we were unable to assess TIV efficacy. This experience illustrates the challenge of conducting a prospective influenza vaccine efficacy trial during a single season when influenza attack rates and drift in circulating strains or B virus lineage match can be difficult to estimate in advance. TRIAL REGISTRATION: Clinical trial registery: NCT00197223.

In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses.

The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.

Protective and cross-protective mucosal immunization of mice by influenza virus type A with bacterial adjuvant.

Mucosal immunization by inactivated viruses often fails to evoke a sufficient immune response. Intensive efforts have been made to enhance the response by suitable adjuvants. We used the G+ nonpathogenic delipidated bacterium Bacillus firmus with pronounced immunostimulatory properties as an adjuvant for immunizing mice with inactivated influenza virus type A. BALB/c mice were immunized intratracheally with inactivated influenza A H1N1 and H3N2 viruses. The production of antibodies in sera and secretions was determined by the ELISA. The local situation in the lungs was assessed histologically and by testing the cytokine expression. The protective and cross-protective effect against infection was tested in in vivo experiments after infection with influenza virus A H1N1. B. firmus as adjuvant increased both systemic and mucosal antibody responses, improved protection against homologous virus and induced cross-protection against virus H1N1 after immunization with virus H3N2.

Correction: MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

[This corrects the article DOI: 10.1371/journal.pone.0151204.].

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay.

Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates.

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay.

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence.

Large-scale nucleotide sequence alignment and sequence variability assessment to identify the evolutionarily highly conserved regions for universal screening PCR assay design: an example of influenza A virus.

The development of a diagnostic polymerase chain reaction (PCR) or quantitative PCR (qPCR) assay for universal detection of highly variable viral genomes is always a difficult task. The purpose of this chapter is to provide a guideline on how to align, process, and evaluate a huge set of homologous nucleotide sequences in order to reveal the evolutionarily most conserved positions suitable for universal qPCR primer and hybridization probe design. Attention is paid to the quantification and clear graphical visualization of the sequence variability at each position of the alignment. In addition, specific problems related to the processing of the extremely large sequence pool are highlighted. All of these steps are performed using an ordinary desktop computer without the need for extensive mathematical or computational skills.

Local-scale diversity and between-year "frozen evolution" of avian influenza A viruses in nature.

Influenza A virus (IAV) in wild bird reservoir hosts is characterized by the perpetuation in a plethora of subtype and genotype constellations. Multiyear monitoring studies carried out during the last two decades worldwide have provided a large body of knowledge regarding the ecology of IAV in wild birds. Nevertheless, other issues of avian IAV evolution have not been fully elucidated, such as the complexity and dynamics of genetic interactions between the co-circulating IAV genomes taking place at a local-scale level or the phenomenon of frozen evolution. We investigated the IAV diversity in a mallard population residing in a single pond in the Czech Republic. Despite the relative small number of samples collected, remarkable heterogeneity was revealed with four different IAV subtype combinations, H6N2, H6N9, H11N2, and H11N9, and six genomic constellations in co-circulation. Moreover, the H6, H11, and N2 segments belonged to two distinguishable sub-lineages. A reconstruction of the pattern of genetic reassortment revealed direct parent-progeny relationships between the H6N2, H11N9 and H6N9 viruses. Interestingly the IAV, with the H6N9 subtype, was re-detected a year later in a genetically unchanged form in the close proximity of the original sampling locality. The almost absolute nucleotide sequence identity of all the respective genomic segments between the two H6N9 viruses indicates frozen evolution as a result of prolonged conservation in the environment. The persistence of the H6N9 IAV in various abiotic and biotic environmental components was also discussed.

Adjuvant effect of Bacillus firmus on the expression of cytokines and toll-like receptors in mouse nasopharynx-associated lymphoid tissue (NALT) after intranasal immunization with inactivated influenza virus type A.

Due to the persisting threat of development of new highly pathogenic influenza A subtypes, a mucosal vaccination which would induce a potent and cross-protective reaction is desirable. We succeeded in mucosal immunization of mice with an inactivated influenza A virus by using delipidated Bacillus firmus (DBF) as adjuvant. The mechanism of adjuvant effect was followed in NALT by comparing the response after intranasal immunization by inactivated influenza virus type A (H1N1) alone, adjuvant alone (DBF), or by a mixture of virus+DBF. Expression of selected gene groups was tested via qPCR at 7 different time-points: cytokines (IL-2, IFN-γ, IL-4, IL-6, and IL-10), type I interferons (IFN-α4, IFN-α11, IFN-α12, and IFN-ß), toll-like receptors (TLR2, TLR3, TLR7, and TLR9), iNOS and CCR7. Intranasally administered DBF and the mixture of virus+DBF induced an elevated expression of IFN-γ, IL-6 and IL-10 cytokines, type I interferons, iNOS, and pDC markers in NALT. Multimarker qPCR data was analyzed by relative quantification and by principal component analysis. DBF has been shown to be a very efficient adjuvant for the stimulation of innate immunity after IN immunization. DBF accelerated, increased, and prolonged the antiviral response.