Molecular characterization of influenza virus circulating in Nepal in the year 2019.

Influenza (sometimes referred to as "flu") is a contagious viral infection of the airways in the lungs that affects a significant portion of the world's population. Clinical symptoms of influenza virus infections can range widely, from severe pneumonia to moderate or even asymptomatic sickness. If left untreated, influenza can have more severe effects on the heart, brain, and lungs than on the respiratory tract and can necessitate hospitalization. This study was aimed to investigate and characterize all types of influenza cases prevailing in Nepal and to analyze seasonal occurrence of Influenza in Nepal in the year 2019. A cross sectional, retrospective and descriptive study was carried out at National Influenza Center (NIC), National Public Health Laboratory Kathmandu Nepal for the period of one year (Jan-Dec 2019). A total of 3606 throat swab samples from various age groups and sexes were processed at the NIC. The specimens were primarily stored at 4 °C and processed using ABI 7500 RT PCR system for the identification of Influenza virus types and subtypes. Data accessed for research purpose were retrieved from National Influenza Centre (NIC) on 1st Jan 2020. Of the total 3606 patients suspected of having influenza infection, influenza viruses were isolated from 1213 (33.6%) patients with male predominance. The highest number of infection was caused by Influenza A/Pdm09 strain 739 (60.9%) followed by Influenza B 304 (25.1%) and Influenza A/H3 169 (13.9%) and most remarkable finding of this study was the detection of H5N1 in human which is the first ever case of such infection in human from Nepal. Similar to other tropical nations, influenza viruses were detected year-round in various geographical locations of Nepal. The influenza virus type and subtypes that were in circulation in Nepal were comparable to vaccine candidate viruses, which the currently available influenza vaccine may prevent.

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.

BACKGROUND: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). RESULTS: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. CONCLUSIONS: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Influenza Virus Genomic Surveillance, Arizona, USA, 2023-2024.

Influenza viruses are constantly evolving and are therefore monitored worldwide in the hope to reduce the burden of disease by annual updates to vaccine recommendations. We conducted genomic sequencing of 110 influenza A and 30 influenza B viruses from specimens collected between October 2023 and February 2024 in Arizona, USA. We identified mutations in the hemagglutinin (HA) antigenic sites as well as the neuraminidase (NA) gene in our samples. We also found no unique HA and NA mutations in vaccinated yet influenza-infected individuals. Real-time genomic sequencing surveillance is important to ensure influenza vaccine effectiveness.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Evaluating an extraction-free sample preparation method for multiplex detection of SARS-Cov-2, influenza A/B, and RSV with implementation on a microfluidic chip.

Following the relaxation of COVID-19 restrictions, other respiratory viruses such as influenza and respiratory syncytial virus (RSV), whose transmission were decreased due to COVID-19 precautions, are rising again. Because of similar clinical features and reported co-infections, multiplex detection of SARS-CoV-2, influenza A/B, and RSV is required to use specific treatments. This study assessed an extraction-free sample preparation (heat treatment at 95°C for 3 minutes) for multiplex detection using rRT-PCR. Despite an observed Ct-delay (∆Ct) averageing 1.26 compared to the standard method, an acceptable total sensitivity of 92 % and a negative predictive value (NPV) of 96 % were obtained. Moreover, Implementation on a microfluidic chip demonstrated efficiency, maintaining an excellent correlation (R2=0.983) with the standard method. Combining this extraction-free procedure with rRT-PCR on a microfluidic chip seems promising, because it simplifies the design and reduces the cost and complexity of the integrated assay for multiplex detection of SARS-CoV-2, influenza A/B, and RSV.

Evaluation of the analytical and clinical performance of two RT-PCR based point-of-care tests; Cepheid Xpert® Xpress CoV-2/Flu/RSV plus and SD BioSensor STANDARD™ M10 Flu/RSV/SARS-CoV-2.

BACKGROUND: Rapid and accurate detection of viral respiratory infections is important for infection control measures. This study compares the analytical and clinical performance of the Xpert® Xpress CoV-2/Flu/RSV plus test ("Xpert", Cepheid) and the STANDARD™ M10 Flu/RSV/SARS-CoV-2 test ("M10", SD Biosensor). Both tests are quadruplex RT-PCR assays for rapid diagnosis of SARS-CoV-2, influenza A/B and RSV. STUDY DESIGN: Analytical sensitivities were determined by limit of detection for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Additionally, the clinical performance of the Xpert and the M10 tests was evaluated against standard-of-care RT-PCR by testing of 492 clinical specimens. RESULTS: The analytical sensitivities for Xpert versus M10 test was 10, 50, 50 and 300 versus 300, 200, 800 and 1500 copies/mL for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Clinical sensitivity for the Xpert test was superior across all four pathogens compared to the M10 test. Xpert showed clinical sensitivity of 100 % in all Ct-ranges for all four pathogens whereas M10 showed clinical sensitivity of 100 % in the 25-30 Ct-range, 84-100 % in the 30-35 Ct-range and 47-67 % in the >35 Ct-range across the four pathogens. Translating into real-life clinical sensitivity, the Xpert would detect 100 % of all four pathogens, whereas M10 would detect 92.1, 92.4, 84.8 and 94.7 % for SARS-CoV-2, influenza A, influenza B and RSV. CONCLUSION: This study demonstrates improved analytical and clinical performance of Xpert Xpress CoV-2/Flu/RSV plus compared to STANDARD M10 Flu/RSV/SARS-CoV-2, which is important for ensuring accuracy of diagnosis at all stages of a respiratory infection.

A live attenuated influenza B virus vaccine expressing RBD elicits protective immunity against SARS-CoV-2 in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to human health globally. It is crucial to develop a vaccine to reduce the effect of the virus on public health, economy, and society and regulate the transmission of SARS-CoV-2. Influenza B virus (IBV) can be used as a vector that does not rely on the current circulating influenza A strains. In this study, we constructed an IBV-based vector vaccine by inserting a receptor-binding domain (RBD) into a non-structural protein 1 (NS1)-truncated gene (rIBV-NS110-RBD). Subsequently, we assessed its safety, immunogenicity, and protective efficacy against SARS-CoV-2 in mice, and observed that it was safe in a mouse model. Intranasal administration of a recombinant rIBV-NS110-RBD vaccine induced high levels of SARS-CoV-2-specific IgA and IgG antibodies and T cell-mediated immunity in mice. Administering two doses of the intranasal rIBV-NS110-RBD vaccine significantly reduced the viral load and lung damage in mice. This novel IBV-based vaccine offers a novel approach for controlling the SARS-CoV-2 pandemic.

Evaluation of Multiplex Rapid Antigen Test for the Detection of SARS-CoV-2 and Influenza A/B in Respiratory Samples.

BACKGROUND: There is little data about the performance of multiplex rapid antigen tests (RATs) on the detection of SARS-CoV-2, influenza A (Flu A), and influenza B (Flu B). This study is to evaluate the performance of Panbio COVID-19/Flu A&B rapid panel (Abbott Diagnostics, Korea) and analyze the factors influencing its sensitivity. METHODS: Nasopharyngeal swabs were collected and stored at the Korea University Anam hospital. In total, 400 residual samples from nasopharyngeal swabs were examined. The diagnostic accuracy of RAT was compared to that of RT-qPCR using the Allplex SARS-CoV-2/FluA/FluB/RSV Assay (Seegene, Seoul, South Korea). RESULTS: Panbio COVID-19/Flu A&B rapid panel showed the sensitivities of 88.0%, 92.0%, and 100% for SARS-CoV-2, Flu A, and Flu B, respectively, and specificities of 100% for all. The agreements with previously licensed single-plex RATs were shown to be high. In the analysis of variables affecting sensitivity, inappropriate sampling time after symptom onset (STASO) and high cycle threshold (Ct value) were shown to negatively affect the sensi-tivity. CONCLUSIONS: In conclusion, the multiplex RAT is useful for diagnosing SARS-CoV-2 and Flu A/B, but more clinical studies are needed.

Molecular Determinants of Drug Resistance and Mutation Patterns in Influenza Viruses Circulating in Poland Across Multiple Epidemic Seasons: Implications for Vaccination Strategies.

BACKGROUND According to the WHO, up to 650 000 people die each year from seasonal flu-related respiratory illnesses. The most effective method of fighting the virus is seasonal vaccination. However, if an infection does occur, antiviral medications should be used as soon as possible. No studies of drug resistance in influenza viruses circulating in Poland have been systematically conducted. Therefore, the aim of the present study was to investigate the drug resistance and genetic diversity of influenza virus strains circulating in Poland by determining the presence of mutations in the neuraminidase gene. MATERIAL AND METHODS A total of 258 clinical specimens were collected during the 2016-2017, 2017-2018, and 2018-2019 epidemic seasons. The samples containing influenza A and B were analyzed by RT-PCR and Sanger sequencing. RESULTS Differences were found between the influenza virus strains detected in different epidemic seasons, demonstrating the occurrence of mutations. Influenza A virus was found to be more genetically variable than influenza B virus (P<0.001, Kruskal-Wallis test). However, there was no significant difference in the resistance prevalence between the influenza A subtypes A/H1N1/pdm09 (4.8%) and A/H3N2/ (6.1%). In contrast, more mutations of drug-resistance genes were found in the influenza B virus (P<0.001, chi-square test). In addition, resistance mutations appeared en masse in vaccine strains circulating in unvaccinated populations. CONCLUSIONS It seems important to determine whether the influenza virus strains tested for drug resistance as part of global influenza surveillance are equally representative of viruses circulating in populations with high and low vaccination rates, for all countries. Our results suggest that countries with low levels of influenza immunization may constitute reservoirs of drug-resistant influenza viruses.

Accurate detection of influenza A virus by use of a novel cross-priming isothermal amplification-based point-of-care assay.

Influenza virus is known to cause respiratory tract infections of varying severity in individuals of all ages. The EasyNAT Rapid Flu assay is a newly developed in vitro diagnostic test that employs cross-priming isothermal amplification (CPA) to detect and differentiate influenza A and B viruses in human nasopharyngeal (NP) swabs. The aim of this study is to determine the performance characteristics of the EasyNAT Rapid Flu assay for rapid detection of influenza virus. The limit of detection (LOD) and cross-reactivity of the EasyNAT Rapid Flu assay were assessed. The clinical performance of the assay was evaluated using NP swab samples that were tested with real-time reverse-transcription polymerase chain reaction (RT-PCR) and Xpert Xpress Flu/RSV assay. The LOD for the detection of influenza A and B using the EasyNAT Rapid Flu assay was found to be 500 copies/mL. Furthermore, the assay exhibited no cross-reactivity with other common respiratory viruses tested. For the 114 NP swab samples tested for influenza A using both the EasyNAT Rapid Flu assay and real-time RT-PCR, the two assays demonstrated a high level of agreement (κ = 0.963, P < 0.001), with a positive percentage agreement (PPA) of 97.7% and a negative percentage agreement (NPA) of 98.6%. Similarly, for the 43 NP swab samples tested for influenza A and B using both the EasyNAT Rapid Flu assay and Xpert Xpress Flu/RSV assay, the two assays showed a high level of agreement (κ = 0.933, P < 0.001), with the overall rate of agreement (ORA) of 97.7% for influenza A and 100% for influenza B. The EasyNAT Rapid Flu assay demonstrates excellent performance in the detection of influenza A, highlighted by its strong agreement with RT-PCR-based assays.IMPORTANCEThe newly developed EasyNAT Rapid Flu assay is an innovative cross-priming isothermal amplification-based method designed for detecting influenza A and B viruses at point-of-care settings. This study aims to thoroughly assess the analytical and clinical performance of the assay, offering valuable insights into its potential advantages and limitations. The findings of this research hold significant implications for clinical practice.

A cost-consequence analysis of the Xpert Xpress CoV-2/Flu/RSV plus test strategy for the diagnosis of influenza-like illnesses.

AIMS: Influenza-like illnesses (ILI) affect millions each year in the United States (US). Determining definitively the cause of symptoms is important for patient management. Xpert Xpress CoV-2/Flu/RSV plus (Xpert Xpress) is a rapid, point-of-care (POC), multiplex real-time polymerase chain reaction (RT-PCR) test intended for the simultaneous qualitative detection and differentiation of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV). The objective of our analysis was to develop a cost-consequence model (CCM) demonstrating the clinico-economic impacts of implementing PCR testing with Xpert Xpress compared to current testing strategies. METHODS: A decision tree model, with a 1-year time horizon, was used to compare testing with Xpert Xpress alone to antigen POC testing and send-out PCR strategies in the US outpatient setting from a payer perspective. A hypothetical cohort of 1,000,000 members was modeled, a portion of whom develop symptomatic ILIs and present to an outpatient care facility. Our main outcome measure is cost per correct treatment course. RESULTS: The total cost per correct treatment course was $1,131 for the Xpert Xpress strategy compared with a range of $3,560 to $5,449 in comparators. POC antigen testing strategies cost more, on average, than PCR strategies. LIMITATIONS: Simplifying model assumptions were used to allow for modeling ease. In clinical practice, treatment options, costs, and diagnostic test sensitivity and specificity may differ from what is included in the model. Additionally, the most recent incidence and prevalence data was used within the model, which is not reflective of historical averages due to the SARS-CoV-2 pandemic. CONCLUSION: The Xpert Xpress CoV-2/Flu/RSV plus test allows for rapid and accurate diagnostic results, leading to reductions in testing costs and downstream healthcare resource utilization compared to other testing strategies. Compared to POC antigen testing strategies, PCR strategies were more efficient due to improved diagnostic accuracy and reduced use of confirmatory testing.

Characterization of an influenza B virus isolated from a fatal case of myocarditis in a pediatric patient in Italy.

Influenza B is one of the infective agents that can cause rapid and fatal myocarditis in children. Here, we describe a fatal case of myocarditis in a previously healthy child, after infection with an influenza B/Victoria-lineage virus during the 2022-23 epidemic season in Italy. Influenza B virus was isolated also in a second case, a younger family member showing only a mild influenza-like illness. Genotypic and phenotypic analyses have been performed on both virus samples and results showed that HA1 sequences were identical and genetically and antigenically related to other B viruses circulating in 2022-23 season in Italy. However, a D129N substitution was found in the receptor binding domain of the HA of the two viruses, not detected in other circulating viruses in Italy but only in a proportion of those circulating in other European countries. Phenotypic analyses assessed the susceptibility towards either neuraminidase inhibitors and baloxavir. Annual influenza vaccination remains one of the best interventions to prevent complications such as myocarditis, particularly in children.

Evaluation of the fully automated, sample-to-result Seegene STARlet-AIOS platform for detection of SARS-CoV-2, influenza virus A, influenza virus B, and RSV.

Early, accurate, and bulk detection of respiratory pathogens is essential for patient management and infection control. STARlet-All-in-One System (AIOS) (Seegene) is a new, fully automated, sample-to-result, molecular diagnostic platform. This study describes the first evaluation of STARlet-AIOS, by testing the Allplex™ SARS-CoV-2 (AS) and Allplex™ SARS-CoV-2/FluA/FluB/RSV combination (AC) assays in comparison to the SARS-CoV-2 assays used at our institute. Over a 3-week period, all naso-/oropharyngeal specimens tested for SARS-CoV-2 using either GeneXpert, Panther, or in-house developed test (LDT) were tested on the AIOS using the AS or AC assays. In addition, retrospective cohorts of specimens containing SARS-CoV-2, influenza virus A, influenza virus B, and RSV were tested. Discrepant results were re-tested with another assay used in this study. Hands-on time (HOT) and turn-around time (TAT) of the different systems were monitored and compared. A total of 738 specimens were tested on the AIOS using the AS assay. In addition, 210 specimens were tested using the AC assay. Overall agreement for SARS-CoV-2 detection was established as 98.5% and 95.2% for the AS and AC assay, respectively. Retrospective testing revealed high agreements for all targets, except for influenza virus A (agreement of 87.5%). HOT of the system was comparable to the HOT of GeneXpert and Panther and TAT comparable to Panther and LDT. The AIOS proved to be a robust sample-to-result system with low HOT and moderate TAT. This study showed reliable detection of SARS-CoV-2, influenza virus B, and RSV, whereas detection of influenza virus A using the AC assay appeared to be suboptimal.

Use of Reverse Genetics for the Generation of Recombinant Influenza Viruses Carrying Nanoluciferase.

Influenza A (FLUAV) and influenza B (FLUBV) viruses are human and/or animal pathogens widely studied due to their importance to public health and animal production. Both FLUAV and FLUBV possess a genome composed of eight viral gene segments. For reverse genetics of influenza viruses, transcription of the mRNA for the viral proteins is typically done from a plasmid encoding an RNA polymerase II (pol II) promoter element upstream of cloned viral cDNA and expressed like host mRNA. On the other side, the synthesis of the negative-sense, single-stranded, uncapped vRNAs can be accomplished by the host's RNA polymerase I (pol I). The reverse genetics for influenza has allowed the manipulation of influenza genomes incorporating heterogeneous sequences into different segments of the influenza genome, such as reporter genes. In this chapter, we outline the protocol from the generation of reverse genetic plasmid that can be applied for the cloning of any of the segments of FLUAV or FLUBV. Furthermore, we describe a protocol for generating FLUAV or FLUBV recombinant viruses carrying Nanoluciferase (NLuc) in the PB1 gene using reverse genetics. Finally, we delineate a microneutralization protocol using FLUAV-NLuc or FLUBV-NLuc viruses optimized for the use of antibodies from different sources (mice, ferrets, avian, etc.), which provides a more sensitive, reliable, and avidity-independent method to assess the presence of neutralizing antibodies against FLUAV or FLUBV.

Persistent predominance of the Victoria lineage of influenza B virus during COVID-19 epidemic in Nanchang, China.

The sentinel hospital-based influenza-like illness (ILI) surveillance network was established in China since the 2009 H1N1 pandemic. This network plays important roles in monitoring influenza virus variation and identifying novel respiratory pathogens. In this study, we characterized the pathogen spectrum pattern (PSP) of ILI based on three sentinel hospitals and analyzed the significant change of PSP during the COVID-19 epidemic. The notable change of influenza virus spectrum was observed since the beginning of COVID-19 outbreak, and we found persistent domination of Victoria lineage of influenza B virus and "extinction" of A/H1N1, A/H3N2, and B/Yamagata during the dynamic Zero-COVID-19 pandemic in Nanchang, China. However, these strains intermittently co-circulated before the COVID-19.

Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A/B viruses.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Influenza A, and Influenza B viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during "flu season" in the post-pandemic era. So far, several multiplex methods have been approved for the simultaneous detection of SARS-CoV-2, Influenza A, and Influenza B. However, due to the rapid mutation rate of the SARS-CoV-2 genome and the emergence of new variants, existing methods must be improved and updated. METHODS: To identify a highly conserved region in the SARS-CoV-2 N-gene, a genomic survey was performed to increase the sensitivity and specificity of primer and probe sets targeting the SARS-CoV-2 genome. The 95% LLOD (95% lower limits of detection) were calculated by probit analysis. A total of 70 predetermined clinical samples using singleplex RT-qPCR assays, were included. The clinical performance of the multiplex RT-qPCR assay was determined and compared with a commercial multiplex kit. The Cohen's kappa coefficient, P-value (McNemar's test), Passing-Bablok regression, and Bland Altman agreement analysis were determined to monitor the agreement of the assays. RESULTS: The novel SARS-CoV-2 primer and probe set designed in this assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical and clinical performance. The 95% LLOD for the multiplex RT-qPCR was 20 copies per reaction for the N gene of SARS-CoV-2, 2 copies per reaction for M1 gene of Influenza A and NS1 gene of Influenza B. The diagnostic sensitivity of the multiplex RT-qPCR was 94.4%, 93.7%, and 100% for the detection of SARS-CoV-2, Influenza A, and Influenza B genomes, respectively. Moreover, the specificity was identical (100%) in both assays. According to the agreement analysis results, there was no statistical difference between our multiplex assay and the commercial kit. CONCLUSIONS: In this study, we developed a novel in-house made multiplex RT-qPCR assay, with high sensitivity, specificity, and reliability for the diagnosis of SARS-CoV-2 infection in clinical samples. This is valuable during Influenza seasons when influenza co-circulates with SARS-CoV-2, as it saves costs, time, and thus specific and timely treatment of patients.

The Influenza B Virus Victoria and Yamagata Lineages Display Distinct Cell Tropism and Infection-Induced Host Gene Expression in Human Nasal Epithelial Cell Cultures.

Understanding Influenza B virus infections is of critical importance in our efforts to control severe influenza and influenza-related diseases. Until 2020, two genetic lineages of influenza B virus-Yamagata and Victoria-circulated in the population. These lineages are antigenically distinct, but the differences in virus replication or the induction of host cell responses after infection have not been carefully studied. Recent IBV clinical isolates of both lineages were obtained from influenza surveillance efforts of the Johns Hopkins Center of Excellence in Influenza Research and Response and characterized in vitro. B/Victoria and B/Yamagata clinical isolates were recognized less efficiently by serum from influenza-vaccinated individuals in comparison to the vaccine strains. B/Victoria lineages formed smaller plaques on MDCK cells compared to B/Yamagata, but infectious virus production in primary human nasal epithelial cell (hNEC) cultures showed no differences. While ciliated epithelial cells were the dominant cell type infected by both lineages, B/Victoria lineages had a slight preference for MUC5AC-positive cells, and B/Yamagata lineages infected more basal cells. Finally, while both lineages induced a strong interferon response 48 h after infection of hNEC cultures, the B/Victoria lineages showed a much stronger induction of interferon-related signaling pathways compared to B/Yamagata. This demonstrates that the two influenza B virus lineages differ not only in their antigenic structure but also in their ability to induce host innate immune responses.

The global epidemic trend analysis of influenza type B drug resistance sites from 2006 to 2018.

INTRODUCTION: Influenza is a severe respiratory viral infection that causes significant morbidity and mortality, due to annual epidemics and unpredictable pandemics. With the extensive use of neuraminidase inhibitor (NAI) drugs, the influenza B virus has carried different drug-resistant mutations. Thus, this study aimed to analyze the prevalence of drug-resistant mutations of the influenza B virus. METHODOLOGY: Near full-length sequences of the neuraminidase (NA) region of all influenza B viruses from January 1, 2006, to December 31, 2018, were downloaded from public databases GISAID and NCBI. Multiple sequence alignments were performed using Clustal Omega 1.2.4 software. Subsequently, phylogenetic trees were constructed by FastTree 2.1.11 and clustered by ClusterPickergui_1.2.3.JAR. Then, the major drug resistance sites and surrounding auxiliary sites were analyzed by Mega-X and Weblogo tools. RESULTS: Among the amino acid sequences of NA from 2006 to 2018, only Clust04 in 2018 carried a D197N mutation of the NA active site, while other drug resistance sites were conserved without mutation. According to the Weblogo analysis, a large number of N198, S295, K373, and K375 mutations were found in the amino acid residues at the auxiliary sites surrounding D197, N294, and R374. CONCLUSIONS: We found the D197N mutation in Clust04 of the 2018 influenza B virus, with a large number of N198, S295, K373, and K375 mutations in the helper sites around N197, N294, and R374 from 2006 to 2018. NA inhibitors are currently the only kind of specific antiviral agent for the influenza B virus, although these mutations cause mild NAIs resistance.

Influenza vaccine effectiveness in preventing hospital encounters for laboratory-confirmed infection among Italian adults, 2022/23 season.

The effectiveness of seasonal influenza vaccination (SIV) varies from year to year. Interim estimates of vaccine effectiveness (VE) in outpatient settings have suggested that the 2022/23 northern hemisphere SIV was 54 % effective. The main goal of this study was to measure the 2022/23 SIV VE among Italian adults in a hospital setting. The study adopted a retrospective test-negative case-control design and was conducted in a large tertiary hospital (Genoa, Italy) between October 2022 and April 2023. Adult (≥18 years) patients accessing the hospital's Emergency Department for symptoms ascribable to an acute respiratory infection, for which a reverse-transcription real-time polymerase chain reaction test for the detection of influenza virus was prescribed, were potentially eligible. Of 33,692 referrals assessed, 487 patients were included in the study. A total of 13 % of patients were positive for influenza, most of which (63 %) belonged to the A(H3N2) subtype. SIV VE was 57 % (95 % CI: 11-81 %), 53 % (95 % CI: 2-80 %) and 38 % (95 % CI: -34-74 %) against any influenza, influenza A and A(H3N2), respectively. Although no cases caused by A(H1N1)pdm09 and B strains were observed among vaccinated individuals, VE estimates against the latter were imprecise, owing to their low detection rates. In conclusion, the 2022/23 SIV was moderately effective against hospital encounters for laboratory-confirmed influenza.

Comparison of two point-of-care respiratory panels for the detection of influenza A/B virus.

BACKGROUND: Rapid and accurate diagnostics of patients with suspected seasonal influenza or pathogens of the upper respiratory tract is crucial. Fast detection is important especially for influenza A/B virus, so that isolation measures should be taken to prevent the spread of the virus. METHODS: We compared the performance of two syndromic testing methodologies (QIAstat-Dx RP, BioFire RP2plus) against the Alere™ i as the comparator method. Totally, 97 swab samples were included from patients with symptoms of acute respiratory infection admitted in the hospitals of the wider region of Crete, Greece. RESULTS: The Positive Percent Agreement (PPA) of the BioFire RP2plus was 100% (95% CI 87.66%-100%), while the Negative Percent Agreement (NPA) was estimated at 91.3% (95% CI 82.03%-96.74%). This method produced no invalid results. For QIAstat-Dx RP the PPA was 89.29% (95% CI 71.77%-97.73%), while the NPA was 91.3% (95% CI 82.03%-96.74%, 63/69). The BioFire RP2plus managed to determine the subtype in more samples than the QIAstat-Dx RP. CONCLUSIONS: Both panels can be valuable tools for clinicians, since they both display high sensitivity and specificity. We report a slightly better performance for BioFire RP2plus, since it produced no invalid results.