RESUMEN

The epidemic and outbreaks of influenza B Victoria lineage (Bv) during 2019-2022 led to an analysis of genetic, epitopes, charged amino acids and Bv outbreaks. Based on the National Influenza Surveillance Network (NISN), the Bv 72 strains isolated during 2019-2022 were selected by spatio-temporal sampling, then were sequenced. Using the Compare Means, Correlate and Cluster, the outbreak data were analyzed, including the single nucleotide variant (SNV), amino acid (AA), epitope, evolutionary rate (ER), Shannon entropy value (SV), charged amino acid and outbreak. With the emergence of COVID-19, the non-pharmaceutical interventions (NPIs) made Less distant transmission and only Bv outbreak. The 2021-2022 strains in the HA genes were located in the same subset, but were distinct from the 2019-2020 strains (P < 0.001). The codon G → A transition in nucleotide was in the highest ratio but the transversion of C → A and T → A made the most significant contribution to the outbreaks, while the increase in amino acid mutations characterized by polar, acidic and basic signatures played a key role in the Bv epidemic in 2021-2022. Both ER and SV were positively correlated in HA genes (R = 0.690) and NA genes (R = 0.711), respectively, however, the number of mutations in the HA genes was 1.59 times higher than that of the NA gene (2.15/1.36) from the beginning of 2020 to 2022. The positively selective sites 174, 199, 214 and 563 in HA genes and the sites 73 and 384 in NA genes were evolutionarily selected in the 2021-2022 influenza outbreaks. Overall, the prevalent factors related to 2021-2022 influenza outbreaks included epidemic timing, Tv, Ts, Tv/Ts, P137 (B → P), P148 (B → P), P199 (P → A), P212 (P → A), P214 (H → P) and P563 (B → P). The preference of amino acid mutations for charge/pH could influence the epidemic/outbreak trends of infectious diseases. Here was a good model of the evolution of infectious disease pathogens. This study, on account of further exploration of virology, genetics, bioinformatics and outbreak information, might facilitate further understanding of their deep interaction mechanisms in the spread of infectious diseases.

Asunto(s)

Brotes de Enfermedades , Evolución Molecular , Gripe Humana , Mutación , Polimorfismo de Nucleótido Simple , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Gripe Humana/genética , Virus de la Influenza B/genética , Aminoácidos/genética , Epítopos/genética , Filogenia , Sustitución de Aminoácidos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética

RESUMEN

BACKGROUND: Influenza B/Yamagata viruses exhibited weak antigenic selection in recent years, reducing their prevalence over time and requiring no update of the vaccine component since 2015. To date, no B/Yamagata viruses have been isolated or sequenced since March 2020. METHODS: The antibody prevalence against the current B/Yamagata vaccine strain in Italy was investigated: For each influenza season from 2012/2013 to 2021/2022, 100 human serum samples were tested by haemagglutination inhibition (HAI) assay against the vaccine strain B/Phuket/3073/2013. In addition, the sequences of 156 B/Yamagata strains isolated during the influenza surveillance activities were selected for analysis of the haemagglutinin genome segment. RESULTS: About 61.9% of the human samples showed HAI antibodies, and 21.7% had protective antibody levels. The prevalence of antibodies at protective levels in the seasons between the isolation of the strain and its inclusion in the vaccine was between 11% and 25%, with no significant changes observed in subsequent years. A significant increase was observed in the 2020/2021 season, in line with the increase in influenza vaccine uptake during the pandemic. Sequence analysis showed that from 2014/2015 season onward, all B/Yamagata strains circulating in Italy were closely related to the B/Phuket/2013 vaccine strain, showing only limited amino acid variation. CONCLUSIONS: A consistent prevalence of antibodies to the current B/Yamagata vaccine strain in the general population was observed. The prolonged use of a well-matched influenza vaccine and a low antigenic diversity of B/Yamagata viruses may have facilitated a strong reduction in B/Yamagata circulation, potentially contributing to the disappearance of this lineage.

Asunto(s)

Anticuerpos Antivirales , Pruebas de Inhibición de Hemaglutinación , Virus de la Influenza B , Vacunas contra la Influenza , Gripe Humana , Italia/epidemiología , Humanos , Virus de la Influenza B/genética , Virus de la Influenza B/clasificación , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/inmunología , Gripe Humana/epidemiología , Gripe Humana/virología , Anticuerpos Antivirales/sangre , Prevalencia , Vacunas contra la Influenza/inmunología , Estaciones del Año , Filogenia , Persona de Mediana Edad , Femenino , Adulto , Masculino , Adolescente , Adulto Joven , Niño , Anciano , Preescolar

RESUMEN

Influenza virus infection poses a continual menace to public health. Here, we developed soluble trimeric HA ectodomain vaccines by establishing interprotomer disulfide bonds in the stem region, which effectively preserve the native antigenicity of stem epitopes. The stable trimeric H1 ectodomain proteins exhibited higher thermal stabilities in comparison with unmodified HAs and showed strong binding activities towards a panel of anti-stem cross-reactive antibodies that recognize either interprotomer or intraprotomer epitopes. Negative stain transmission electron microscopy (TEM) analysis revealed the stable trimer architecture of the interprotomer disulfide-stapled WA11#5, NC99#2, and FLD#1 proteins as well as the irregular aggregation of unmodified HA molecules. Immunizations of mice with those trimeric HA ectodomain vaccines formulated with incomplete Freund's adjuvant elicited significantly more potent cross-neutralizing antibody responses and offered broader immuno-protection against lethal infections with heterologous influenza strains compared to unmodified HA proteins. Additionally, the findings of our study indicate that elevated levels of HA stem-specific antibody responses correlate with strengthened cross-protections. Our design strategy has proven effective in trimerizing HA ectodomains derived from both influenza A and B viruses, thereby providing a valuable reference for designing future influenza HA immunogens.

Asunto(s)

Anticuerpos Neutralizantes , Anticuerpos Antivirales , Disulfuros , Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas contra la Influenza , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae , Animales , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Anticuerpos Antivirales/inmunología , Ratones , Disulfuros/química , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Anticuerpos Neutralizantes/inmunología , Femenino , Protección Cruzada/inmunología , Reacciones Cruzadas , Humanos , Gripe Humana/prevención & control , Gripe Humana/inmunología , Gripe Humana/virología , Epítopos/inmunología , Epítopos/genética , Epítopos/química , Multimerización de Proteína , Virus de la Influenza B/inmunología , Virus de la Influenza B/genética , Virus de la Influenza B/química

RESUMEN

The COVID-19 pandemic, in addition to the co-occurrence of influenza virus and respiratory syncytial virus (RSV), has emphasized the requirement for efficient and reliable multiplex diagnostic methods for respiratory infections. While existing multiplex detection techniques are based on reverse transcription quantitative polymerase chain reaction (RT-qPCR) and extraction and purification kits, the need for complex instrumentation and elevated cost limit their scalability and availability. In this study, we have developed a point-of-care (POC) device based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that can simultaneously detect four respiratory viruses (SARS-CoV-2, Influenza A, Influenza B, and RSV) and perform two controls in less than 30 min, while avoiding the use of the RNA extraction kit. The system includes a disposable microfluidic cartridge with mechanical components that automate sample processing, with a low-cost and portable optical reader and a smartphone app to record and analyze fluorescent images. The application as a real point-of-care platform was validated using swabs spiked with virus particles in nasal fluid. Our portable diagnostic system accurately detects viral RNA specific to respiratory pathogens, enabling deconvolution of coinfection information. The detection limits for each virus were determined using virus particles spiked in chemical lysis buffer. Our POC device has the potential to be adapted for the detection of new pathogens and a wide range of viruses by modifying the primer sequences. This work highlights an alternative approach for multiple respiratory virus diagnostics that is well-suited for healthcare systems in resource-limited settings or at home.

Asunto(s)

Técnicas de Amplificación de Ácido Nucleico , Sistemas de Atención de Punto , SARS-CoV-2 , Humanos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , COVID-19/diagnóstico , COVID-19/virología , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , ARN Viral/análisis , ARN Viral/aislamiento & purificación , ARN Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Virus Sincitiales Respiratorios/aislamiento & purificación , Virus Sincitiales Respiratorios/genética

RESUMEN

We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.

Asunto(s)

Antígenos Virales , Hurones , Pruebas de Inhibición de Hemaglutinación , Vacunas contra la Influenza , Animales , Perros , Vacunas contra la Influenza/inmunología , Células de Riñón Canino Madin Darby , Pruebas de Inhibición de Hemaglutinación/métodos , Antígenos Virales/inmunología , Humanos , Chlorocebus aethiops , Anticuerpos Antivirales/inmunología , Pruebas de Neutralización , Células Vero , Cultivo de Virus/métodos , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/prevención & control , Gripe Humana/inmunología , Gripe Humana/virología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/genética , Línea Celular , Virus de la Influenza B/inmunología , Virus de la Influenza B/genética

RESUMEN

Influenza is a highly contagious acute viral illness that affects the respiratory system, posing a significant global public health concern. Influenza B virus (IBV) causes annual seasonal epidemics. The exploration of molecular biology and reverse genetics of IBV is pivotal for understanding its replication, pathogenesis, and evolution. Reverse genetics empowers us to purposefully alter the viral genome, engineer precise genetic modifications, and unveil the secrets of virulence and resistance mechanisms. It helps us in quickly analyzing new virus strains by viral genome manipulation and the development of innovative influenza vaccines. Reverse genetics has been employed to create mutant or reassortant influenza viruses for evaluating their virulence, pathogenicity, host range, and transmissibility. Without this technique, these tasks would be difficult or impossible, making it crucial for preparing for epidemics and protecting public health. Here, we bring together the latest information on how we can manipulate the genes of the influenza B virus using reverse genetics methods, most importantly helper virus-independent techniques.

Asunto(s)

Virus de la Influenza B , Vacunas contra la Influenza , Gripe Humana , Genética Inversa , Virus de la Influenza B/genética , Virus de la Influenza B/inmunología , Genética Inversa/métodos , Humanos , Gripe Humana/prevención & control , Gripe Humana/epidemiología , Gripe Humana/virología , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Genoma Viral , Animales , Desarrollo de Vacunas , Biología Molecular/métodos , Virulencia/genética , Epidemias/prevención & control

RESUMEN

Influenza virus is a well-known pathogen that can cause epidemics and pandemics. Several surveillance methods are being followed to monitor the transmission patterns and spread of influenza in the community. Wastewater-based Epidemiology (WBE) can serve as an additional tool to detect the presence of influenza viruses. The current study primarily focuses on surveillance of Influenza A and Influenza B in wastewater treatment plant (WWTP) samples. A total of 100 wastewater samples were collected in July (n = 50) and August (n = 50) 2023 from four different WWTPs in Manipal and Udupi, district of Karnataka, India. The WWTP samples were processed and tested by Real-Time reverse transcriptase PCR (RT-PCR). The data generated was analysed in comparison with the clinical Influenza cases. Of the 100 samples, 18 (18%) tested positive for Influenza A virus and 2 (2%) tested positive for Influenza B virus, with a viral load ranging 1.4 x 102-2.2 x 103 gc/L for influenza A virus and 5.2 x 103-7.7 x 103gc/L for influenza B virus. On correlating the WWTP positivity with clinical case, it was found that influenza clinical cases and virus positivity in wastewater increased simultaneously, emphasizing WBE as a concurrent method for monitoring influenza virus activity.

Asunto(s)

Hospitales , Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Aguas Residuales , Aguas Residuales/virología , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Humanos , India/epidemiología , Gripe Humana/virología , Gripe Humana/epidemiología , Purificación del Agua , Carga Viral , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Introduction: Seasonal influenza generally represents an underestimated public health problem with significant socioeconomic implications. Monitoring and detecting influenza epidemics are important tasks that require integrated strategies. Wastewater-based epidemiology (WBE) is an emerging field that uses wastewater data to monitor the spread of disease and assess the health of a community. It can represent an integrative surveillance tool for better understanding the epidemiology of influenza and prevention strategies in public health. Methods: We conducted a study that detected the presence of Influenza virus RNA using a wastewater-based approach. Samples were collected from five wastewater treatment plants in five different municipalities, serving a cumulative population of 555,673 Sicilian inhabitants in Italy. We used the RT-qPCR test to compare the combined weekly average of Influenza A and B viral RNA in wastewater samples with the average weekly incidence of Influenza-like illness (ILI) obtained from the Italian national Influenza surveillance system. We also compared the number of positive Influenza swabs with the viral RNA loads detected from wastewater. Our study investigated 189 wastewater samples. Results: Cumulative ILI cases substantially overlapped with the Influenza RNA load from wastewater samples. Influenza viral RNA trends in wastewater samples were similar to the rise of ILI cases in the population. Therefore, wastewater surveillance confirmed the co-circulation of Influenza A and B viruses during the season 2022/2023, with a similar trend to that reported for the weekly clinically confirmed cases. Conclusion: Wastewater-based epidemiology does not replace traditional epidemiological surveillance methods, such as laboratory testing of samples from infected individuals. However, it can be a valuable complement to obtaining additional information on the incidence of influenza in the population and preventing its spread.

Asunto(s)

Virus de la Influenza A , Gripe Humana , Aguas Residuales , Sicilia/epidemiología , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Aguas Residuales/virología , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Estaciones del Año , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , ARN Viral/análisis , Ciudades/epidemiología

RESUMEN

The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.

Asunto(s)

COVID-19 , Virus de la Influenza A , Virus de la Influenza B , Reacción en Cadena de la Polimerasa Multiplex , Ribonucleasa P , SARS-CoV-2 , Humanos , Ribonucleasa P/genética , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , COVID-19/diagnóstico , COVID-19/virología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Diagnóstico Diferencial , Gripe Humana/diagnóstico , Gripe Humana/virología , Sensibilidad y Especificidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Límite de Detección , ARN Viral/genética , ARN Viral/análisis

RESUMEN

BACKGROUND: Non-pharmaceutical measures and travel restrictions have halted the spread of coronavirus disease 2019 (COVID-19) and influenza. Nonetheless, with COVID-19 restrictions lifted, an unanticipated outbreak of the influenza B/Victoria virus in late 2021 and another influenza H3N2 outbreak in mid-2022 occurred in Guangdong, southern China. The mechanism underlying this phenomenon remains unknown. To better prepare for potential influenza outbreaks during COVID-19 pandemic, we studied the molecular epidemiology and phylogenetics of influenza A(H3N2) and B/Victoria that circulated during the COVID-19 pandemic in this region. METHODS: From January 1, 2018 to December 31, 2022, we collected throat swabs from 173,401 patients in Guangdong who had acute respiratory tract infections. Influenza viruses in the samples were tested using reverse transcription-polymerase chain reaction, followed by subtype identification and sequencing of hemagglutinin (HA) and neuraminidase (NA) genes. Phylogenetic and genetic diversity analyses were performed on both genes from 403 samples. A rigorous molecular clock was aligned with the phylogenetic tree to measure the rate of viral evolution and the root-to-tip distance within strains in different years was assessed using regression curve models to determine the correlation. RESULTS: During the early period of COVID-19 control, various influenza viruses were nearly undetectable in respiratory specimens. When control measures were relaxed in January 2020, the influenza infection rate peaked at 4.94% (39/789) in December 2021, with the influenza B/Victoria accounting for 87.18% (34/39) of the total influenza cases. Six months later, the influenza infection rate again increased and peaked at 11.34% (255/2248) in June 2022; influenza A/H3N2 accounted for 94.51% (241/255) of the total influenza cases in autumn 2022. The diverse geographic distribution of HA genes of B/Victoria and A/H3N2 had drastically reduced, and most strains originated from China. The rate of B/Victoria HA evolution (3.11 × 10-3, P < 0.05) was 1.7 times faster than before the COVID-19 outbreak (1.80 × 10-3, P < 0.05). Likewise, the H3N2 HA gene's evolution rate was 7.96 × 10-3 (P < 0.05), which is 2.1 times faster than the strains' pre-COVID-19 evolution rate (3.81 × 10-3, P < 0.05). CONCLUSIONS: Despite the extraordinarily low detection rate of influenza infection, concealed influenza transmission may occur between individuals during strict COVID-19 control. This ultimately leads to the accumulation of viral mutations and accelerated evolution of H3N2 and B/Victoria viruses. Monitoring the evolution of influenza may provide insights and alerts regarding potential epidemics in the future.

Asunto(s)

COVID-19 , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Epidemiología Molecular , Filogenia , SARS-CoV-2 , Humanos , COVID-19/epidemiología , COVID-19/virología , COVID-19/transmisión , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , China/epidemiología , Gripe Humana/epidemiología , Gripe Humana/virología , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/clasificación , SARS-CoV-2/genética , Adulto , Persona de Mediana Edad , Masculino , Femenino , Pandemias , Adulto Joven , Anciano , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Adolescente , Neuraminidasa/genética , Niño , Preescolar

RESUMEN

Influenza, a human disease caused by viruses in the Orthomyxoviridae family, is estimated to infect 5% -10 % of adults and 20% -30 % of children annually. Influenza A (IAV) and Influenza B (IBV) viruses accumulate amino acid substitutions (AAS) in the hemagglutinin (HA) and neuraminidase (NA) proteins seasonally. These changes, as well as the dominating viral subtypes, vary depending on geographical location, which may impact disease prevalence and the severity of the season. Genomic surveillance is crucial for capturing circulation patterns and characterizing AAS that may affect disease outcomes, vaccine efficacy, or antiviral drug activities. In this study, whole-genome sequencing of IAV and IBV was attempted on positive remnant clinical samples (587) collected from 580 patients between June 2023 and February 2024 in the Johns Hopkins Health System (JHHS). Full-length HA segments were obtained from 424 (72.2 %) samples. H1N1pdm09 (71.7 %) was the predominant IAV subtype, followed by H3N2 (16.7 %) and IBV-Victoria clade V1A.3a.2 (11.6 %). Within H1N1pdm09 HA sequences, the 6B1A.5a.2a.1 (60.5 %) clade was the most represented. Full-length NA segments were obtained from 421 (71.7 %) samples. Within H1N1pdm09 and IBV, AAS previously proposed to change susceptibility to NA inhibitors were infrequently detected. Phylogeny of HA and NA demonstrated heterogeneous HA and NA H1N1pdm09 and IBV subclades. No significant differences were observed in admission rates or use of supplemental oxygen between different subtypes or clades. Influenza virus genomic surveillance is essential for understanding the seasonal evolution of influenza viruses and their association with disease prevalence and outcomes.

Asunto(s)

Evolución Molecular , Genoma Viral , Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Neuraminidasa , Filogenia , Estaciones del Año , Secuenciación Completa del Genoma , Humanos , Gripe Humana/virología , Gripe Humana/epidemiología , Adulto , Virus de la Influenza B/genética , Virus de la Influenza B/clasificación , Virus de la Influenza A/genética , Virus de la Influenza A/clasificación , Neuraminidasa/genética , Persona de Mediana Edad , Masculino , Adulto Joven , Femenino , Adolescente , Niño , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Preescolar , Anciano , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Lactante , Sustitución de Aminoácidos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Anciano de 80 o más Años

RESUMEN

Influenza vaccination is the most cost-effective strategy for influenza prevention. Influenza vaccines have been found to be effective against symptomatic and medically attended outpatient influenza illnesses. However, there is currently a lack of data regarding the effectiveness of inactivated influenza vaccines in Chongqing, China. We conducted a prospective observational test-negative design study. Outpatient and emergency cases presenting with influenza-like illnesses (ILI) and available influenza reverse transcription polymerase chain reaction (RT-PCR) were selected and classified as cases (positive influenza RT-PCR) or controls (negative influenza RT-PCR). A total of 7,307 cases of influenza and 7,905 control subjects were included in this study. The overall adjusted influenza vaccine effectiveness (IVE) was 44.4% (95% confidence interval (CI): 32.5-54.2%). In the age groups of less than 6 years old, 6-18 years old, and 19-59 years old, the adjusted IVE were 32.2% (95% CI: 10.0-48.9%), 48.2% (95% CI: 30.6-61.4%), and 72.0% (95% CI: 43.6-86.1%). The adjusted IVE for H1N1, H3N2 and B (Victoria) were 71.1% (95% CI: 55.4-81.3%), 36.1% (95% CI: 14.6-52.2%) and 33.7% (95% CI: 14.6-48.5%). Influenza vaccination was effective in Chongqing from 2018 to 2022. Evaluating IVE in this area is feasible and should be conducted annually in the future.

Asunto(s)

Vacunas contra la Influenza , Gripe Humana , Eficacia de las Vacunas , Humanos , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , China/epidemiología , Adolescente , Adulto , Gripe Humana/prevención & control , Persona de Mediana Edad , Adulto Joven , Niño , Masculino , Femenino , Preescolar , Estudios Prospectivos , Lactante , Anciano , Vacunación/estadística & datos numéricos , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Anciano de 80 o más Años , Virus de la Influenza B/inmunología , Virus de la Influenza B/genética

RESUMEN

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 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) 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.

Asunto(s)

Variación Genética , Virus de la Influenza B , Gripe Humana , Neuraminidasa , Filogenia , SARS-CoV-2 , Humanos , Gripe Humana/virología , Gripe Humana/epidemiología , Virus de la Influenza B/genética , Virus de la Influenza B/clasificación , Virus de la Influenza B/aislamiento & purificación , SARS-CoV-2/genética , SARS-CoV-2/clasificación , Neuraminidasa/genética , Adulto , Masculino , Persona de Mediana Edad , Femenino , Bulgaria/epidemiología , Adulto Joven , Anciano , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Preescolar , Niño , Adolescente , COVID-19/epidemiología , COVID-19/virología , Lactante , Estaciones del Año , Virus de la Influenza A/genética , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/clasificación , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación

RESUMEN

The M2 proteins of influenza A and B viruses form acid-activated proton channels that are essential for the virus lifecycle. Proton selectivity is achieved by a transmembrane (TM) histidine whereas gating is achieved by a tryptophan residue. Although this functional apparatus is conserved between AM2 and BM2 channels, AM2 conducts protons exclusively inward whereas BM2 conducts protons in either direction depending on the pH gradient. Previous studies showed that in AM2, mutations of D44 abolished inward rectification of AM2, suggesting that the tryptophan gate is destabilized. To elucidate how charged residues C-terminal to the tryptophan regulates channel gating, here we investigate the structure and dynamics of H19 and W23 in a BM2 mutant, GDR-BM2, in which three BM2 residues are mutated to the corresponding AM2 residues, S16G, G26D and H27R. Whole-cell electrophysiological data show that GDR-BM2 conducts protons with inward rectification, identical to wild-type (WT) AM2 but different from WT-BM2. Solid-state NMR 15N and 13C spectra of H19 indicate that the mutant BM2 channel contains higher populations of cationic histidine and neutral τ tautomers compared to WT-BM2 at acidic pH. Moreover, 19F NMR spectra of 5-19F-labeled W23 resolve three peaks at acidic pH, suggesting three tryptophan sidechain conformations. Comparison of these spectra with the tryptophan spectra of other M2 peptides suggests that these indole sidechain conformations arise from interactions with the C-terminal charged residues and with the N-terminal cationic histidine. Taken together, these solid-state NMR data show that inward rectification in M2 proton channels is accomplished by tryptophan interactions with charged residues on both its C-terminal and N-terminal sides. Gating of these M2 proton channels is thus accomplished by a multi-residue complex with finely tuned electrostatic and aromatic interactions.

Asunto(s)

Histidina , Virus de la Influenza B , Protones , Triptófano , Proteínas de la Matriz Viral , Triptófano/química , Histidina/química , Histidina/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Matriz Viral/genética , Virus de la Influenza B/química , Virus de la Influenza B/genética , Virus de la Influenza A/química , Virus de la Influenza A/metabolismo , Virus de la Influenza A/genética , Concentración de Iones de Hidrógeno , Canales Iónicos/química , Canales Iónicos/metabolismo , Canales Iónicos/genética , Mutación , Simulación de Dinámica Molecular , Proteínas Viroporinas

RESUMEN

Background: Accurate detection of influenza virus in clinical samples requires correct execution of all aspects of the detection test. If the viral load in a sample is below the detection limit, a false negative result may be obtained. To overcome this issue, we developed a modified transport medium (MTM) for clinical sample transportation to increase viral detection sensitivity. Method: We first validated the MTM using laboratory-stocked influenza A viruses (IAVs: H1N1, H3N2, H7N3, H9N2) and influenza B viruses (IBVs: Yamagata, Victoria). We also tested clinical samples. A total of 110 patients were enrolled and a pair of samples were collected to determine the sensitivity of real-time polymerase chain reaction (RT-PCR) following MTM treatment. Result: After 24 h culturing in MTM, the viral loads were increased, represented by a 10-fold increase in detection sensitivity for H1N1, H9N2, and IBVs, a 100-fold increase for H3N2, and a 1,000-fold increase for H7N3. We further tested the effects of MTM on 19 IAV and 11 IBV stored clinical samples. The RT-PCR results showed that the positive detection rate of IAV samples increased from 63.16% (12/19) without MTM culturing to 78.95% (15/19) after 48 h culturing, and finally 89.47% (17/19) after 72 h culturing. MTM treatment of IBV clinical samples also increased the positive detection rate from 36.36% (4/11, 0 h) to 63.64% (7/11, 48 h) to 72.73% (8/11, 72 h). For clinical samples detected by RT-PCR, MTM outperformed other transport mediums in terms of viral detection rate (11.81% increase, P=0.007). Conclusion: Our results demonstrated that the use of MTM for clinical applications can increase detection sensitivity, thus facilitating the accurate diagnosis of influenza infection.

Asunto(s)

Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Sensibilidad y Especificidad , Manejo de Especímenes , Carga Viral , Humanos , Gripe Humana/diagnóstico , Gripe Humana/virología , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , Manejo de Especímenes/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Medios de Cultivo/química , Persona de Mediana Edad , Femenino , Adulto , Masculino

RESUMEN

Respiratory tract infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses are persistent and critical. The Cobas Liat SARS-CoV-2 & influenza A/B assay (Multiplex Liat), the FDA-authorized point-of-care reverse transcriptase polymerase chain reaction (RT-PCR) assay, has a turnaround time of 20 min and high accuracy. This study evaluates the pooled performance of this assay to provide practical information. This meta-analysis was registered in PROSPERO (registration number: CRD42023467579). A systematic literature search was conducted within PubMed, Ovid-EMBASE, and the Cochrane Library for articles evaluating the accuracy of the Multiplex Liat assay through September 2023. A random-effects model was used to calculate the pooled diagnostic values with real-time RT-PCR (rRT-PCR) as a reference test. A total of 4,705 samples from eight studies were included in the primary meta-analysis. The overall pooled sensitivity and specificity of Multiplex Liat were 100.0 % (95 % confidence interval [CI] = 96.7 %-100.0 %) and 99.7 % (95 % CI = 98.7 %-99.9 %), respectively. The presence of variants of concern or in-house rRT-PCR assays as reference standards did not significantly affect the pooled diagnostic performance of the Multiplex Liat. When 5,333 samples from nine studies were assessed for sensitivity, the pooled sensitivity was 100.0 % (95 % CI = 85.8 %-100.0 %) without a significant difference. This meta-analysis demonstrates the usefulness of Multiplex Liat for the detection of SARS-CoV-2 based on pooled diagnostic values. These practical findings may facilitate appropriate settings for the diagnosis and management of patients with respiratory tract infections.

Asunto(s)

COVID-19 , Virus de la Influenza A , Gripe Humana , SARS-CoV-2 , Sensibilidad y Especificidad , Humanos , Gripe Humana/diagnóstico , Gripe Humana/virología , COVID-19/diagnóstico , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , Prueba de Ácido Nucleico para COVID-19/métodos , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos

RESUMEN

During the COVID-19 pandemic, non-pharmaceutical interventions were introduced to reduce exposure to respiratory viruses. However, these measures may have led to an "immunity debt" that could make the population more vulnerable. The goal of this study was to examine the transmission dynamics of seasonal influenza in the years 2023-2024. Respiratory samples from patients with influenza-like illness were collected and tested for influenza A and B viruses. The electronic medical records of index cases from October 2023 to March 2024 were analyzed to determine their clinical and epidemiological characteristics. A total of 48984 positive cases were detected, with a pooled prevalence of 46.9% (95% CI 46.3-47.5). This season saw bimodal peaks of influenza activity, with influenza A peaked in week 48, 2023, and influenza B peaked in week 1, 2024. The pooled positive rates were 28.6% (95% CI 55.4-59.6) and 18.3% (95% CI 18.0-18.7) for influenza A and B viruses, respectively. The median values of instantaneous reproduction number were 5.5 (IQR 3.0-6.7) and 4.6 (IQR 2.4-5.5), respectively. The hospitalization rate for influenza A virus (2.2%, 95% CI 2.0-2.5) was significantly higher than that of influenza B virus (1.1%, 95% CI 0.9-1.4). Among the 17 clinical symptoms studied, odds ratios of 15 symptoms were below 1 when comparing influenza A and B positive inpatients, with headache, weakness, and myalgia showing significant differences. This study provides an overview of influenza dynamics and clinical symptoms, highlighting the importance for individuals to receive an annual influenza vaccine.

Asunto(s)

Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Estaciones del Año , Humanos , Gripe Humana/epidemiología , Masculino , Femenino , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , Adulto , Persona de Mediana Edad , Adolescente , Adulto Joven , Niño , Anciano , Preescolar , Beijing/epidemiología , Lactante , COVID-19/epidemiología , COVID-19/transmisión , Prevalencia , Recién Nacido , Susceptibilidad a Enfermedades , Anciano de 80 o más Años , SARS-CoV-2

RESUMEN

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.

Asunto(s)

COVID-19 , Coinfección , Filogenia , SARS-CoV-2 , Humanos , Coinfección/virología , Coinfección/epidemiología , SARS-CoV-2/genética , SARS-CoV-2/clasificación , SARS-CoV-2/aislamiento & purificación , COVID-19/virología , COVID-19/epidemiología , Persona de Mediana Edad , Adulto , Femenino , Masculino , Adolescente , Preescolar , Niño , Anciano , Adulto Joven , Lactante , Infecciones del Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/epidemiología , Rhinovirus/genética , Rhinovirus/clasificación , Rhinovirus/aislamiento & purificación , Virus de la Influenza A/genética , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Virus Sincitial Respiratorio Humano/clasificación , Nasofaringe/virología , Secuenciación Completa del Genoma , China/epidemiología , Estaciones del Año , Anciano de 80 o más Años , Genoma Viral , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/clasificación

RESUMEN

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1-5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2-5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

Asunto(s)

Coinfección , Virus de la Influenza B , Gripe Humana , Filogenia , Virus Reordenados , Virus Reordenados/genética , Virus Reordenados/aislamiento & purificación , Virus Reordenados/clasificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/clasificación , Humanos , Niño , Gripe Humana/virología , Coinfección/virología , Genoma Viral , Masculino , Proteínas Virales/genética

RESUMEN

BACKGROUND: The concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting. METHODS: Two time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2. RESULTS: The results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022. CONCLUSION: The results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected.

Asunto(s)

COVID-19 , Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , SARS-CoV-2 , Humanos , COVID-19/epidemiología , COVID-19/diagnóstico , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/genética , Gripe Humana/epidemiología , Gripe Humana/diagnóstico , Gripe Humana/virología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Virus Sincitial Respiratorio Humano/genética , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/genética , Masculino , Femenino , Coinfección/epidemiología , Coinfección/diagnóstico , Persona de Mediana Edad , Adulto , Técnicas de Diagnóstico Molecular/métodos , Estaciones del Año , Anciano