Heterogeneity of Circulating Influenza Viruses and Their Impact on Influenza Virus Vaccine Effectiveness During the Influenza Seasons 2016/17 to 2018/19 in Austria.

The constantly changing pattern in the dominance of viral strains and their evolving subclades during the seasons substantially influences influenza vaccine effectiveness (IVE). In order to further substantiate the importance of detailed data of genetic virus characterization for IVE estimates during the seasons, we performed influenza virus type and subtype specific IVE estimates. IVE estimates were assessed using a test-negative case-control design, in the context of the intraseasonal changes of the heterogeneous mix of circulating influenza virus strains for three influenza seasons (2016/17 to 2018/19) in Austria. Adjusted overall IVE over the three seasons 2016/17, 2017/18, and 2018/19 were -26, 39, and 63%, respectively. In accordance with the changing pattern of the circulating strains a broad range of overall and subtype specific IVEs was obtained: A(H3N2) specific IVE ranged between -26% for season 2016/17 to 58% in season 2018/19, A(H1N1)pdm09 specific IVE was 25% for the season 2017/18 and 65% for the season 2018/19 and Influenza B specific IVE for season 2017/18 was 45%. The results obtained in our study over the three seasons demonstrate the increasingly complex dynamic of the ever changing genetic pattern of the circulating influenza viruses and their influence on IVE estimates. This emphasizes the importance of detailed genetic virus surveillance for reliable IVE estimates.

Distinct differences in clinical manifestation and viral laboratory parameters between children and adults with influenza A(H1N1)pdm09 infection--a retrospective comparative analysis.

During the influenza pandemic 2009 children and adults differed in the clinical course of the influenza disease. In following the question arose, if the case definitions used within the national and international organizations are an adequate tool for the clinical diagnosis of influenza in children as well as in adults. Therefore medical charts from 146 children and 229 adults were retrospectively analyzed. In addition, the initial viral loads of all 375 patients and the duration of virus shedding of a subset of 79 patients were also investigated. Children show a wider clinical spectrum including gastro enteric symptoms and also a different spectrum of laboratory parameters like elevated CRP-levels, leucocytosis, and higher viral loads. Further, children show significantly more often complications, for example, myositis that may be underdiagnosed. In patients receiving antiviral-therapy complications occurred significantly less often and the presence of symptoms was significantly shorter compared to the untreated group (2.3 days vs. 6.0 days). In summary, the differences in the clinical picture between children and adults should be taken into consideration for the clinical diagnosis of influenza and also for a future discussion on age specific influenza case definitions.

Attributable deaths due to influenza: a comparative study of seasonal and pandemic influenza.

Influenza epidemics lead to an increase in hospitalizations and deaths. Up to now the overall impact of attributable deaths due to seasonal and pandemic influenza viruses in Austria has not been investigated in detail. Therefore we compared the number and age distribution of influenza associated deaths during ten influenza epidemic seasons to those observed during the pandemic influenza A(H1N1)2009 season. A Poisson model, relating age and daily deaths to week of influenza season using national mortality and viral surveillance data adjusted for the confounding effect of co-circulating Respiratory Syncytial Virus was used. We estimated an average of 316 influenza associated deaths per seasonal influenza epidemic (1999/2000-2008/2009) and 264 for the pandemic influenza season 2009/2010 in the area of Vienna, Austria. Comparing the mortality data for seasonal and pandemic influenza viruses in different age groups revealed a statistically significant increase in mortality for pandemic A(H1N1)2009 influenza virus in the age groups below 34 years of age and a significant decrease in mortality in those above 55 years. Our data adjusted for co-circulating RSV confirm the different mortality pattern of seasonal and pandemic influenza A(H1N1)2009 virus in different age groups.

Differentiating inherited human herpesvirus type 6 genome from primary human herpesvirus type 6 infection by means of dried blood spot from the newborn screening card.

To differentiate active human herpesvirus type 6 (HHV-6) infection from inherited HHV-6 (iHHV-6), we analyzed dried blood spots from archived newborn screening cards in 3 patients with high HHV-6 DNA copy numbers. Two patients were positive for HHV-6 DNA as neonates suggesting iHHV-6. In 1 patient, the absence of HHV-6 DNA excluded iHHV-6.

Diagnosis of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections in all age groups often requiring hospitalization. Rapid laboratory diagnosis of RSV infection significantly decreases the use of antibiotics, additional laboratory testing and is associated with shorter hospitalization periods. The specific diagnosis of RSV infection is based on the detection of virus or viral antigens or virus specific nucleic acid sequences in respiratory secretions. The kind and quality of the clinical specimen exerts a considerable influence on the results of all currently used viral detection assays. Antigen based tests are widely available, easy to perform and the results are available in a short time but their reduced sensitivity and specificity represent a considerable shortcoming. Among the methods available isolation in cell culture was considered the gold standard for the sensitive identification of RSV but is gradually replaced by highly sensitive and specific nucleic acid amplification assays that provide more rapid results. Of these reverse transcription polymerase chain reaction (PCR) was the first and is still the most frequently used nucleic acid-based assay. New methodologies, as for example the real-time PCR methods allow the quantification of viral nucleic acids in the clinical sample. Disadvantages of the nucleic acid based assays are their high costs and their limited standardization.Future research on new methodologies for the diagnosis of viral respiratory tract infections should focus on the development of sensitive, rapid and cost effective test systems allowing the screening for all probable causative agents. In addition varying testing protocols for summer and winter months based on epidemiologic data are needed to direct their practical use.

Human metapneumovirus subgroup changes and seasonality during epidemics.

BACKGROUND: Human metapneumovirus (HMPV) is a major cause of respiratory tract illness in young children and causes annual outbreaks in winter and spring seasons. We evaluated the subgroups of HMPV that caused annual outbreaks and its seasonal occurrence during a 21-year period. METHODS: Real-time PCR was used for detection of HMPV in 3576 nasopharyngeal aspirates that had been continuously collected year-round for the years 1987 to 2008 from infants hospitalized with acute respiratory tract illness. Phylogenetic analysis was used to assess HMPV subgroups. RESULTS: Of the 3576 samples obtained, 202 (5.6%) tested positive for HMPV. All known HMPV subgroups (A1, A2a, A2b, B1, B2) could be identified as important respiratory tract pathogens in infants. We found that one HMPV subgroup predominated each year, and it was displaced by another subgroup every 1 to 3 years. Besides the frequent change in predominant HMPV subgroups, we observed a yearly shift in the seasonal occurrence, with a strong peak of HMPV activity in late spring-summer months every second year. CONCLUSION: HMPV activity is characterized by a periodic change in the predominant subgroup and it shows a stable seasonal rhythm of alternating winter and spring activity.

A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1.

BACKGROUND. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-H1N1 vaccine was well tolerated. Rhinitis-like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. CONCLUSIONS. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. TRIAL REGISTRATION. ClinicalTrials.gov identifier: NCT00724997 .

Influenza B mutant viruses with truncated NS1 proteins grow efficiently in Vero cells and are immunogenic in mice.

Contemporary influenza B virus strains were generated encoding C-terminally truncated NS1 proteins. Viable viruses containing the N-terminal 14, 38, 57 or 80 aa of the NS1 protein were rescued in Vero cells. The influenza B virus NS1-truncated mutants were impaired in their ability to counteract interferon (IFN) production, induce antiviral pro-inflammatory cytokines early after infection and show attenuated or restricted growth in IFN-competent hosts. In Vero cells, all of the mutant viruses replicated to high titres comparable to the wild-type influenza B virus. Mice that received a single, intranasal immunization of the NS1-truncated mutants elicited an antibody response and protection against wild-type virus challenge. Therefore, these NS1-truncated mutants should prove useful as potential candidates for live-attenuated influenza virus vaccines.

Biennial spring activity of human metapneumovirus in Austria.

BACKGROUND: Human metapneumovirus (HMPV) is considered an important respiratory pathogen in young children. To gain insight into the seasonality and epidemiologic characteristics of HMPV infection, this study determined the frequency of HMPV infections in hospitalized infants during a 7-year period. METHODS: By use of real-time reverse-transcriptase polymerase chain reaction, nasopharyngeal aspirates from 1612 infants less than 2 years of age who were hospitalized for acute respiratory tract illness were tested for the presence of HMPV. Weekly HMPV testing data were analyzed to assess the timing of HMPV activity. Season variability was estimated by comparing the onset, duration, peak, and end of outbreaks from October 2000 through October 2007. RESULTS: Overall, 109 (6.8%) of 1612 cases of acute respiratory illness were associated with HMPV infection. Seasonal HMPV activity varied substantially from year to year, both in prevalence rates of HMPV cases and in seasonal timing of outbreaks. HMPV activity was characterized by a biennial rhythm, with spring seasons occurring every second year, and these accounted for a substantial proportion, up to 30%, of hospitalized cases of acute respiratory tract illness. CONCLUSIONS: HMPV activity varies substantially from year to year, both in the frequency and timing of illness and shows a biennial pattern of alternating winter and spring activity.

Dynamics of antigenic and genetic changes in the hemagglutinins of influenza A/H3N2 viruses of three consecutive seasons (2002/2003 to 2004/2005) in Austria.

Human influenza viruses are subject to continuous antigenic drift and this phenomenon poses great problems for the annual production of vaccines which should ideally be manufactured from strains closely matching the predominant strains of the coming influenza season. We have investigated the dynamics of antigenic and genetic changes in the hemagglutinins of circulating influenza A/H3N2 strains in three consecutive seasons (2002/2003 to 2004/2005) in Austria by sequence analysis of the HA1 domain and by antigenic characterization using a hemagglutination inhibition assay. Each of the three seasons was dominated by a single and different H3N2 variant, but in all cases sequencing revealed the co-circulation of a drift variant which would have been missed by conventional antigenic analysis. These emerging strains always showed already a close genetic relationship to the dominating strain of the following season. Our results underscore the value of monitoring seasonal influenza strain dynamics by sequence analysis as an instrument that can provide important and timely information on the appearance of strains with epidemiologic significance.

Emergence of multiple cytomegalovirus strains in blood and lung of lung transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) is a major pathogen in lung transplant recipients (LTRs). The emergence of different CMV strains in lung and blood after transplantation has not yet been analyzed. METHODS: In total, 75 serum and 91 broncheoalveolar lavage (BAL) samples obtained from 25 LTRs in the follow-up after transplantation were tested for the presence of different CMV strains. The gB, gN, and gO genes of the CMV isolates were analyzed by subtype-specific PCR, restriction fragment length polymorphism (RFLP), sequencing, and phylogenetic analysis. RESULTS: Mixed CMV-strain populations were detected after cessation of antiviral prophylaxis in up to 80% and 90% of the patients' BAL and serum, respectively, and this was independent of the CMV serostatus of donor and recipient. In five patients, the same single CMV strain was consistently detectable over at least 1 year in lung and blood, although in two of these cases donor and recipient had both been CMV-seropositive. Most CMV strains were distributed in the lung and blood compartment. Symptomatic CMV infection within the first year after transplantation was observed only in patients with mixed CMV-strain populations (P<0.05). CONCLUSION: Most LTRs harbor more than one CMV strain in their lung and blood compartment after cessation of prophylaxis, but the CMV strain distribution within and between the compartments varies between individuals and is not associated with the donor/recipient serostatus. The data further show that compartmentalization of CMV strains in lung versus blood seems to be a rare event and that the presence of mixed CMV-strain infections within the first year after transplantation may be disadvantageous for LTRs.

Single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response.

BACKGROUND: Dual respiratory viral infections are frequently associated with lower respiratory tract illness in infants. This study aimed to determine the impact of a dual respiratory viral infection on specific aspects of the infant's immune response and the clinical course of illness. METHODS: A prospective study was performed with 772 infants hospitalized from October 2000 through July 2004. Sensitive polymerase chain reaction methodology revealed the presence of a single respiratory virus in 443 (57%) of 772 cases, whereas dual infections were identified in 153 (20%) of cases. From 250 infants with confirmed respiratory viral infection, fresh heparinized blood was analyzed for interferon-gamma (IFN-gamma) responses by flow cytometry. Of these, 191 patients had a single infection with respiratory syncytial virus (RSV), rhinoviruses, adenoviruses or influenza viruses; and 59 patients had a dual infection with RSV and rhinoviruses, RSV and adenoviruses, influenza viruses and rhinoviruses or adenoviruses and rhinoviruses. The clinical features and peripheral lymphocyte IFN-gamma responses were compared among infants with single or dual infections. RESULTS: It was found that dual infections with non-RSV respiratory viruses induced peripheral blood mononuclear cell IFN-gamma responses that mimic those of single infections, whereas coinfection with RSV was associated with reduced IFN-gamma responses and a more severe clinical course of lower respiratory tract disease. CONCLUSIONS: The results indicate that the clinical characteristics and the IFN-gamma response differ significantly in single and dual respiratory viral infection, depending on the nature of the simultaneously detected viruses. In dual infections, RSV involvement was associated with a decreased IFN-gamma response in peripheral blood mononuclear cell and an increase in severity of illness.

An international external quality assessment of nucleic acid amplification of herpes simplex virus.

BACKGROUND: There is an increasing awareness of the need for external quality control of diagnostic virology. OBJECTIVES: To assess the quality of nucleic acid amplification tests (NAT) of herpes simplex within Europe. STUDY DESIGN: Herpes simplex virus (HSV) proficiency panels were produced at the Swedish Institute for Infectious Disease Control on behalf of the European Union Concerted Action for Quality Control of Nucleic Acid Amplification in 1999 and 2000. Nine reference laboratories evaluated the production process. Each panel consisted of 12 coded samples with various concentrations of inactivated, freeze-dried HSV type 1 (HSV-1), and HSV type 2 (HSV-2), or negative controls. Positive samples included HSV-1 and HSV-2 in a range of concentrations (2 x 10(2) to 2 x 10(7) genome copies per ml) similar to those found in cerebrospinal fluids from patients with HSV encephalitis. RESULTS: Sixty-six participants reported a total of 76 data sets for panel 1, and 71 reported 78 data sets for panel 2. The majority of the participants employed qualitative 'in-house' polymerase chain reaction (PCR) methods, either in a single, nested or semi-nested format. For panel 2, 9 laboratories reported use of 'real-time' PCR in contrast to 3 for panel 1. Three laboratories submitted quantitative results on both panels. Thirty percent of the data sets had correct results for the entire panel 1. In 6 data sets (8%) a total of 11 false positive results were reported. For panel 2, 28% of the data sets had correct result. Nineteen false positive results were reported in 14 data sets (18%), but most of the incorrect results reflected a lack of test sensitivity. CONCLUSIONS: The relatively high frequency of false positive results and the large number of false-negative results, albeit at low copy number, stress the need for improvement in the quality of HSV NAT and for external quality control programmes.

Adenovirus DNA in serum of children hospitalized due to an acute respiratory adenovirus infection.

Serum samples from 68 immunocompetent infants (mean age, 12.6 months) with an acute adenovirus infection of the respiratory tract (39 experiencing their first adenovirus infection) were tested for the presence of adenovirus DNA, to investigate whether viral dissemination via the blood is usually present in the immunocompetent patient. Using a nested polymerase chain reaction assay, adenovirus DNA could be detected in acute-phase serum samples from 28 (41%) children. Adenovirus DNA was never found in follow-up serum samples, indicating a short period ( approximately 1 week) of viral dissemination. In children experiencing their first adenovirus infection, viral DNA could be detected in 72% of the acute-phase serum samples collected within the first week after onset of symptoms. Adenovirus DNA could also be detected in 25% of the acute-phase serum samples from patients with reinfection.