Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Los linajes Victoria y Yamagata de los virus gripales B, desconocidos y poco valorados / The Victoria and Yamagata Lineages of Influenza B Viruses, unknown and undervalued

El virus gripal B pertenece a la familia Orthomyxoviriridae y al género Influenzavirus B. Presenta un genoma de tipoARN negativo formado por unos 14.648 nucleótidos divididosen ocho segmentos distintos que codifican unas 11 proteínas.Antes de 1980 todos los virus de la gripe B pertenecían a unúnico linaje genético; pero en este año emergieron dos linajesantigénica y genéticamente distintos que se denominaron B/Victoria/2/1987 y B/Yamagata/16/1988. Se han podido demostrar procesos de intercambio genético intralinajes y entrelinajes; de ellos los mas frecuentes son aquellos en los que el linajeVictoria adquiere genes del linaje Yamagata. Se ha propuestoque las diferencias en las dinámicas evolutivas de los dos linajesse deban a las diferentes preferencias de unión de la hemaglutinina gripal al receptor celular. El linaje Victoria ha mostradocapacidad para unirse a los receptores celulares con restos deácido siálico en las posiciones a-2,3 y a-2,6; mientras que ellinaje Yamagata lo hace exclusivamente en las posiciones humanas a-2,6 del tracto respiratorio. La escasa circulación enlos últimos meses podría haber contribuido a la eliminación(“extinción”) temporal del linaje Yamagata. Desde 2017 la casitotalidad de las cepas de este linaje pertenecen al clado 3A,cuando con anterioridad se detectaban clados múltiples circulando. Aunque este clado 3A es diverso a nivel genético y haadquirido mutaciones sustitutivas en el gen de la hemaglutinina, éstas no han determinado cambios antigénicos significativos que hayan obligado a sustituir su componente antigénico(B/Pukhet/3073/2013) en la vacuna gripal desde 2015. (AU)

The influenza virus B belongs to the family Orthomyxoviriridae and to the genus Influenzavirus B. It has a negativeRNA-type genome made up of about 14,648 nucleotides divided into eight different segments that encode about 11 proteins.Before 1980 all influenza B viruses belonged to a single geneticlineage; but in this year two antigenically and genetically distinct lineages emerged which were named B/Victoria/2/1987and B/Yamagata/16/1988. Intralineage and interlineage genetic exchange processes have been demonstrated; The most frequent of them are those in which the Victoria lineage acquiresgenes from the Yamagata lineage. It has been proposed thatthe differences in the evolutionary dynamics of the two lineages are due to the different binding preferences of influenzahemagglutinin to the cellular receptor. The Victoria lineage hasshown the ability to bind to cell receptors with sialic acid residues at the α-2,3 and α-2,6 positions; whereas the Yamagatalineage does so exclusively in the human α-2,6 positions of therespiratory tract. Low circulation in recent months may havecontributed to the temporary elimination (“extinction”) of theYamagata lineage. Since 2017, almost all of the strains of thislineage belong to clade 3A, when previously multiple circulating clades were detected. Although this clade 3A is diverse atthe genetic level and has acquired surrogate mutations in thehemagglutinin gene, these have not determined significantantigenic changes that have made it necessary to replace itsantigenic component (B/Pukhet/3073/2013) in the influenzavaccine since 2015. (AU)

Cell-Adapted Mutations and Antigenic Diversity of Influenza B Viruses in Missouri, 2019-2020 Season.

Influenza B viruses (IBVs) are causing an increasing burden of morbidity and mortality, yet the prevalence of culture-adapted mutations in human seasonal IBVs are unclear. We collected 368 clinical samples from patients with influenza-like illness in Missouri during the 2019-2020 influenza season and recovered 146 influenza isolates including 38 IBV isolates. Of MDCK-CCL34, MDCK-Siat1, and humanized MDCK (hCK), hCK showed the highest virus recovery efficiency. All Missourian IBVs belonged to the Victoria V1A.3 lineage, all of which contained a three-amino acid deletion on the HA protein and were antigenically distant from the Victoria lineage IBV vaccine strain used during that season. By comparing genomic sequences of these IBVs in 31 paired samples, eight cell-adapted nonsynonymous mutations were identified, with the majority in the RNA polymerase. Analyses of IBV clinical sample-isolate pairs from public databases further showed that cell- and egg-adapted mutations occurred more widely in viral proteins, including the receptor and antibody binding sites on HA. Our study suggests that hCK is an effective platform for IBV isolation and that culture-adapted mutations may occur during IBV isolation. As culture-adapted mutations may affect subsequent virus studies and vaccine development, the knowledge from this study may help optimize strategies for influenza surveillance, vaccine strain selection, and vaccine development.

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus.

A panel of influenza virus-like sequences were recently documented in fish and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterize the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV. Functionally, we show that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that is sensitive to NA inhibitors. We tested the functionality of the HA by addressing the receptor specificity, stability, preferential airway protease cleavage, and fusogenicity. We show highly specific binding to monosialic ganglioside 2 (GM2) and fusogenicity at a range of different pH conditions. In addition, we found limited antigenic conservation of the WSEIV HA and NA relative to the B/Malaysia/2506/2004 virus HA and NA. In summary, we perform a functional and antigenic characterization of the glycoproteins of WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish.

Lineage-specific protection and immune imprinting shape the age distributions of influenza B cases.

How a history of influenza virus infections contributes to protection is not fully understood, but such protection might explain the contrasting age distributions of cases of the two lineages of influenza B, B/Victoria and B/Yamagata. Fitting a statistical model to those distributions using surveillance data from New Zealand, we found they could be explained by historical changes in lineage frequencies combined with cross-protection between strains of the same lineage. We found additional protection against B/Yamagata in people for whom it was their first influenza B infection, similar to the immune imprinting observed in influenza A. While the data were not informative about B/Victoria imprinting, B/Yamagata imprinting could explain the fewer B/Yamagata than B/Victoria cases in cohorts born in the 1990s and the bimodal age distribution of B/Yamagata cases. Longitudinal studies can test if these forms of protection inferred from historical data extend to more recent strains and other populations.

Differences in the age distribution of influenza B virus infection according to influenza B virus lineages in the Korean population.

OBJECTIVES: Since the 2000s, two lineages of the influenza B virus (influenza B/Victoria and influenza B/Yamagata) have been co-circulating. Information on the age distribution of patients infected by each influenza B virus lineage may be helpful for establishing differentiated influenza prevention and control strategies for each age group. METHODS: Age distributions were compared between patients infected by influenza A and B viruses and between those infected by the influenza B virus when B/Victoria and B/Yamagata lineages circulated dominantly. RESULTS: Between the 2014-2015 and 2018-2019 influenza seasons, 2,718 and 1,397 patients were diagnosed with influenza A and B virus infections, respectively. The median age of patients infected by the influenza B virus was lower than that of patients infected by the influenza A virus (8 vs 12 years, p < 0.001). In the Yamagata season, the median ages of patients infected by influenza A and B viruses were similar (12 vs 11 years, p = 0.732); however, in the Victoria season, the median age of patients infected by the influenza B virus was lower than that of patients infected by the influenza A virus (6 vs 10 years, p < 0.001). In patients infected by the influenza B virus, patients aged <6 years and those aged ≥6 years were more likely to be infected during the Victoria and Yamagata seasons, respectively (p < 0.001). CONCLUSION: The age distribution of patients infected by the influenza virus was different between the Yamagata and Victoria seasons. Different influenza prevention and control strategies should be considered on the basis of the predominantly circulating virus and the affected age group.

Detection and discrimination of influenza B Victoria lineage deletion variant viruses by real-time RT-PCR.

BackgroundDuring the 2016/17 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (aa) deletions in the haemagglutinin (HA) protein. There are currently five antigenically distinct HA proteins expressed by co-circulating influenza B viruses: B/YAM, B/VIC V1A (no deletion), B/VIC V1A-2DEL (2 aa deletion) and two antigenically distinguishable groups of B/VIC V1A-3DEL (3 aa deletion). The prevalence of these viruses differs across geographical regions, making it critical to have a sensitive, rapid diagnostic assay that detects and distinguishes these influenza B variant viruses during surveillance.AimOur objective was to develop a real-time RT-PCR (rRT-PCR) assay for detection and discrimination of influenza B/VIC lineage variant viruses.MethodsWe designed a diagnostic assay with one pair of conserved primers and three probes specific to each genetic group. We used propagated influenza B/VIC variant viruses and clinical specimens to assess assay performance.ResultsThis rRT-PCR assay detects and distinguishes the influenza B/VIC V1A, B/VIC V1A-2DEL, and B/VIC V1A-3DEL variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost.ConclusionCoupling this assay with the Centers for Disease Control and Prevention's Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterisation of circulating influenza B viruses. Detailed surveillance information on these distinct influenza B variant viruses will provide insight into their prevalence and geographical distribution and could aid in vaccine recommendations.

Impact of quadrivalent influenza vaccines in Brazil: a cost-effectiveness analysis using an influenza transmission model.

BACKGROUND: Influenza epidemics significantly weight on the Brazilian healthcare system and its society. Public health authorities have progressively expanded recommendations for vaccination against influenza, particularly to the pediatric population. However, the potential mismatch between the trivalent influenza vaccine (TIV) strains and those circulating during the season remains an issue. Quadrivalent vaccines improves vaccines effectiveness by preventing any potential mismatch on influenza B lineages. METHODS: We evaluate the public health and economic benefits of the switch from TIV to QIV for the pediatric influenza recommendation (6mo-5yo) by using a dynamic epidemiological model able to consider the indirect impact of vaccination. Results of the epidemiological model are then imputed in a health-economic model adapted to the Brazilian context. We perform deterministic and probabilistic sensitivity analysis to account for both epidemiological and economical sources of uncertainty. RESULTS: Our results show that switching from TIV to QIV in the Brazilian pediatric population would prevent 406,600 symptomatic cases, 11,300 hospitalizations and almost 400 deaths by influenza season. This strategy would save 3400 life-years yearly for an incremental direct cost of R$169 million per year, down to R$86 million from a societal perspective. Incremental cost-effectiveness ratios for the switch would be R$49,700 per life-year saved and R$26,800 per quality-adjusted life-year gained from a public payer perspective, and even more cost-effective from a societal perspective. Our results are qualitatively similar in our sensitivity analysis. CONCLUSIONS: Our analysis shows that switching from TIV to QIV to protect children aged 6mo to 5yo in the Brazilian influenza epidemiological context could have a strong public health impact and represent a cost-effective strategy from a public payer perspective, and a highly cost-effective one from a societal perspective.

A six-plex droplet digital RT-PCR assay for seasonal influenza virus typing, subtyping, and lineage determination.

BACKGROUND: There are two influenza A subtypes (H1 and H3) and two influenza B lineages (Victoria and Yamagata) that currently co-circulate in humans. In this study, we report the development of a six-plex droplet digital RT-PCR (ddRT-PCR) assay that can detect HA and M segments of influenza A (H1, H3, and M) and influenza B (Yamagata HA, Victoria HA, and M) viruses in a single reaction mixture. It can simultaneously detect six different nucleic acid targets in a ddRT-PCR platform. METHODS: The six-plex ddRT-PCR used in this study is an amplitude-based multiplex assay. The analytical performance of the assay was evaluated. Correlation with standard qRT-PCR methodology was assessed using 55 clinical samples. RESULTS: The assay has a wide dynamic range, and it has good reproducibility within and between runs. The limit of quantification of each target in this assay ranged from 15 copies/reaction for influenza B Victoria M gene to 45 copies/reaction for influenza B Yamagata M gene. In addition, this assay can accurately quantify each of these targets in samples containing viral RNAs from two different viruses that were mixed in a highly skewed ratio. Typing, subtyping, and lineage differentiation data of 55 tested clinical respiratory specimens were found to be identical to those deduced from standard monoplex qRT-PCR assays. CONCLUSIONS: The six-plex ddRT-PCR test was demonstrated to be highly suitable for detecting dual influenza infection cases. This assay is expected to be a useful diagnostic tool for clinical and research use.

Severity and outcomes of influenza-related pneumonia in type A and B strains in China, 2013-2019.

BACKGROUND: Inconsistencies exist regarding the severity of illness caused by different influenza strains. The aim of this study was to compare the clinical outcomes of hospitalized adults and adolescents with influenza-related pneumonia (Flu-p) from type A and type B strains in China. METHODS: We retrospectively reviewed data from Flu-p patients in five hospitals in China from January 2013 to May 2019. Multivariate logistic and Cox regression models were used to assess the effects of influenza virus subtypes on clinical outcomes, and to explore the risk factors of 30-day mortality for Flu-p patients. RESULTS: In total, 963 laboratory-confirmed influenza A-related pneumonia (FluA-p) and 386 influenza B-related pneumonia (FluB-p) patients were included. Upon adjustment for confounders, multivariate logistic regression models showed that FluA-p was associated with an increased risk of invasive ventilation (adjusted odds ratio [aOR]: 3.824, 95% confidence interval [CI]: 2.279-6.414; P <  0.001), admittance to intensive care unit (aOR: 1.630, 95% CI: 1.074-2.473, P = 0.022) and 30-day mortality (aOR: 2.427, 95% CI: 1.568-3.756, P <  0.001) compared to FluB-p. Multivariate Cox regression models confirmed that influenza A virus infection (hazard ratio: 2.637, 95% CI: 1.134-6.131, P = 0.024) was an independent predictor for 30-day mortality in Flu-p patients. CONCLUSIONS: The severity of illness and clinical outcomes of FluA-p patients are more severe than FluB-p. This highlights the importance of identifying the virus strain during the management of severe influenza.

Treatment-Emergent Influenza Virus Polymerase Acidic Substitutions Independent of Those at I38 Associated With Reduced Baloxavir Susceptibility and Virus Rebound in Trials of Baloxavir Marboxil.

Influenza viruses harboring treatment-emergent I38F/M/N/T substitutions in the polymerase acidic (PA) endonuclease exhibited reduced susceptibility to baloxavir and were associated with virus rebound and variable clinical response in clinical trials. US regulatory review of registrational trial data also identified treatment-emergent PA substitutions E23K in A/H1N1 viruses and E23G/K, A37T, and E199G in A/H3N2 viruses, which conferred reduced susceptibility to baloxavir, although to a lesser degree than I38F/M/N/T substitutions, and were associated with virus rebound. Although these non-I38 substitutions emerged less frequently than substitutions at I38, they represent alternate pathways to baloxavir virologic resistance and should be monitored accordingly.

Epidemiological features and time-series analysis of influenza incidence in urban and rural areas of Shenyang, China, 2010-2018.

In recent years, there have been a significant influenza activity and emerging influenza strains in China, resulting in an increasing number of influenza virus infections and leading to public health concerns. The aims of this study were to identify the epidemiological and aetiological characteristics of influenza and establish seasonal autoregressive integrated moving average (SARIMA) models for forecasting the percentage of visits for influenza-like illness (ILI%) in urban and rural areas of Shenyang. Influenza surveillance data were obtained for ILI cases and influenza virus positivity from 18 sentinel hospitals. The SARIMA models were constructed to predict ILI% for January-December 2019. During 2010-2018, the influenza activity was higher in urban than in rural areas. The age distribution of ILI cases showed the highest rate in young children aged 0-4 years. Seasonal A/H3N2, influenza B virus and pandemic A/H1N1 continuously co-circulated in winter and spring seasons. In addition, the SARIMA (0, 1, 0) (0, 1, 2)12 model for the urban area and the SARIMA (1, 1, 1) (1, 1, 0)12 model for the rural area were appropriate for predicting influenza incidence. Our findings suggested that there were regional and seasonal distinctions of ILI activity in Shenyang. A co-epidemic pattern of influenza strains was evident in terms of seasonal influenza activity. Young children were more susceptible to influenza virus infection than adults. These results provide a reference for future influenza prevention and control strategies in the study area.

Co-circulation of influenza A(H1N1), A(H3N2), B(Yamagata) and B(Victoria) during the 2017-2018 influenza season in Zhejiang Province, China.

Influenza is a major human respiratory pathogen. Due to the high levels of influenza-like illness (ILI) in Zhejiang, China, the control and prevention of influenza was challenging during the 2017-2018 season. To identify the clinical spectrum of illness related to influenza and characterise the circulating influenza virus strains during this period, the characteristics of ILI were studied. Viral sequencing and phylogenetic analyses were conducted to investigate the virus types, substitutions at the amino acid level and phylogenetic relationships between sequences. This study has shown that the 2017/18 influenza season was characterised by the co-circulation of influenza A (H1N1) pdm09, A (H3N2) and B viruses (both Yamagata and Victoria lineage). From week 36 of 2017 to week 12 of 2018, ILI cases accounted for 5.58% of the total number of outpatient and emergency patient visits at the surveillance sites. Several amino acid substitutions were detected. Vaccination mismatch may be a potential reason for the high percentage of ILI. Furthermore, it is likely that multiple viral introductions played a role in the endemic co-circulation of influenza in Zhejiang, China. More detailed information regarding the molecular epidemiology of influenza should be included in long-term influenza surveillance.

Circulation of Influenza Type B Lineages in Greece During 2005-2015 and Estimation of Their Impact.

The past decades influenza B lineages Victoria and Yamagata cocirculated. Our aim was to estimate the distribution of the two lineages circulating in Greece and any possible mismatching with vaccine influenza B strains. We studied 490 laboratory-confirmed influenza B nonsevere acute respiratory infection (non-SARI) cases diagnosed in the two National Influenza Reference Laboratories by reverse transcriptase polymerase chain reaction from July 1, 2005 to June 30, 2015 and 100 influenza B SARI cases diagnosed from July 1, 2011 to June 30, 2015. Median matching between the circulating influenza B lineages and the vaccine influenza B strains was 19.2% (range: 0-100%) for non-SARI cases during 2005-2015 and 67.6% (range: 41.2-94.1%) for SARI cases during 2011-2015. In two influenza seasons (2005-2006 and 2006-2007), complete lineage mismatch between influenza B non-SARI cases and influenza B vaccine strains was found. We estimated that 5, 12, or 16 laboratory-confirmed SARI cases could have been prevented by quadrivalent influenza inactivated vaccine (QIV) during the 2011-2012 season and 1, 2, or 3 SARI cases during the 2014-2015 season, with a vaccination coverage rate of 70% and a vaccine effectiveness of 20%, 50%, or 70%, respectively. Significant cocirculation of Victoria and Yamagata B strains and mismatching with vaccine influenza B strains were found during 2005-2015 in Greece. The wide use of a QIV instead of a TIV will confer additional immunity and therefore protection against influenza B, and it is expected to prevent several SARI cases annually. Our findings strongly support the recommendations for using QIV.

Antigenic variants of influenza B viruses isolated in Japan during the 2017-2018 and 2018-2019 influenza seasons.

BACKGROUND: Here, we genetically and antigenically analyzed influenza B viruses (IBVs) isolated in Japan during the 2017-2018 and 2018-2019 influenza seasons. METHODS: A total of 68 IBVs (61 B/Yamagata/16/88-like [B/Yamagata]-lineage and 7 B/Victoria/2/87-like [B/Victoria]-lineage) were antigenically and genetically characterized by using hemagglutination inhibition (HI) assays and phylogenetic analysis, respectively. The susceptibility of IBVs to neuraminidase (NA) inhibitors was assessed by using a fluorescence-based NA inhibition assay. RESULTS: All 61 B/Yamagata-lineage isolates were genetically closely related to B/Phuket/3073/2013, the vaccine strain for these two seasons. Eleven B/Yamagata-lineage isolates tested were antigenically similar to B/Phuket/3073/2013 by the HI test. Seven B/Victoria-lineage isolates were genetically closely related to B/Texas/02/2013, the WHO-recommended vaccine strain for the 2017-2018 season; however, they were antigenically distinct from B/Texas/02/2013 with an eightfold or 16-fold difference in HI titer. Of these 7 isolates, 4 possessed a two-amino-acid deletion at positions 162 and 163 in hemagglutinin (HA) and the other 3 had a three-amino-acid deletion at positions 162-164 in HA. Importantly, the variants with the three-amino-acid deletion appeared to be antigenically different from the B/Colorado/06/2017 virus with the two-amino-acid deletion, the vaccine strain for the 2018-2019 season with a fourfold or eightfold difference in HI titer. One B/Yamagata-lineage isolate carrying a G407S mutation in its NA showed a marked reduction in susceptibility to zanamivir, peramivir, and laninamivir. CONCLUSIONS: These results highlight the need for continued monitoring for the prevalence of the antigenic variant with the three-amino-acid deletion and the variant with reduced NA inhibitor susceptibility.

Validation of the Seegene RV15 multiplex PCR for the detection of influenza A subtypes and influenza B lineages during national influenza surveillance in hospitalized adults.

Background. The Serious Outcomes Surveillance Network of the Canadian Immunization Research Network (CIRN SOS) has been performing active influenza surveillance since 2009 (ClinicalTrials.gov identifier: NCT01517191). Influenza A and B viruses are identified and characterized using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), and multiplex testing has been performed on a subset of patients to identify other respiratory virus aetiologies. Since both methods can identify influenza A and B, a direct comparison was performed.Methods. Validated real-time RT-PCRs from the World Health Organization (WHO) to identify influenza A and B viruses, characterize influenza A viruses into the H1N1 or H3N2 subtypes and describe influenza B viruses belonging to the Yamagata or Victoria lineages. In a subset of patients, the Seeplex RV15 One-Step ACE Detection assay (RV15) kit was also used for the detection of other respiratory viruses.Results. In total, 1111 nasopharyngeal swabs were tested by RV15 and real-time RT-PCRs for influenza A and B identification and characterization. For influenza A, RV15 showed 98.0 % sensitivity, 100 % specificity and 99.7 % accuracy. The performance characteristics of RV15 were similar for influenza A subtypes H1N1 and H3N2. For influenza B, RV15 had 99.2 % sensitivity, 100 % specificity and 99.8 % accuracy, with similar assay performance being shown for both the Yamagata and Victoria lineages.Conclusions. Overall, the detection of circulating subtypes of influenza A and lineages of influenza B by RV15 was similar to detection by real-time RT-PCR. Multiplex testing with RV15 allows for a more comprehensive respiratory virus surveillance in hospitalized adults, without significantly compromising the reliability of influenza A or B virus detection.

Development and evaluation of a conventional RT-PCR for differentiating emerging influenza B/Victoria lineage viruses with hemagglutinin amino acid deletion from B/Yamagata lineage viruses.

BACKGROUND: Recent influenza B/Victoria lineage viruses contain amino acid deletions at positions 162 to 164 of the haemagglutinin (HA) protein. These amino acid deletions have affected the detection of B/Victoria lineage viruses by the lineage-specific conventional reverse-transcription polymerase chain reaction (RT-PCR) that was recommended by World Health Organization (WHO). OBJECTIVES: We aimed to develop and evaluate a novel lineage-specific RT-PCR for rapid differentiation of the contemporary B/Victoria lineage from B/Yamagata lineage viruses. STUDY DESIGN: Primers of our in-house RT-PCR were designed to avoid amino acid positions 162 to 164 and to target conserved regions of the HA gene that are specific for B/Victoria and B/Yamagata lineage viruses. Our in-house RT-PCR and WHO RT-PCR were evaluated using influenza B positive clinical specimens or virus culture isolates. Influenza B virus lineage was confirmed by Sanger sequencing. RESULTS: A total of 105 clinical specimens or virus culture isolates were retrieved, including 83 with B/Victoria lineage and 22 with B/Yamagata lineage viruses. Our in-house RT-PCR correctly identified B/Victoria lineage viruses in all 83 samples, including 82 samples with double or triple amino acid deletion in the HA protein. Conversely, the WHO lineage-specific conventional RT-PCR failed to detect any of the 82 samples with HA amino acid deletions. For the 22 samples with B/Yamagata lineage viruses, both RT-PCR assays have correctly identified B/Yamagata lineage in all samples. CONCLUSIONS: Our novel lineage-specific RT-PCR has successfully detected all contemporary B/Victoria lineage viruses with amino acid deletions in HA. This protocol is especially useful for laboratories without the equipment for real-time PCR.

Spatial, Temporal and Genetic Dynamics Characteristics of Influenza B Viruses in China, 1973-2018.

Annual influenza B virus epidemics and outbreaks cause severe influenza diseases in humans and pose a threat to public health. China is an important epidemic area of influenza B viruses. However, the spatial, temporal transmission pathways and the demography history of influenza B viruses in China remain unknown. We collected the haemagglutinin gene sequences sampled of influenza B virus in China between 1973 and 2018. A Bayesian Markov chain Monte Carlo phylogeographic discrete approach was used to infer the spatial and temporal phylodynamics of influenza B virus. The Bayesian phylogeographic analysis of influenza B viruses showed that the North subtropical and South subtropical zones are the origins of the Victoria and Yamagata lineage viruses, respectively. Furthermore, the South temperate and North subtropical zones acted as transition nodes in the Victoria lineage virus dispersion network and that the North subtropical and Mid subtropical zones acted as transition nodes in the Yamagata lineage virus dispersion network. Our findings contribute to the knowledge regarding the spatial and temporal patterns of influenza B virus outbreaks in China.

Debate on the compositions of influenza B in northern hemisphere seasonal influenza vaccines.

Background: Annual influenza vaccination is the most effective way to prevent influenza. Influenza vaccines have traditionally included the hemagglutinins (HA) and neuraminidases (NA) from the two A viruses (H1N1 and H3N2) and either B Yamagata or B Victoria. Mismatches between circulating isolates of influenza B and the vaccines are very common. Taking 2017/2018 winter in northern hemisphere as an example, this study was designed to find out the reasons for mismatch between the trivalent influenza vaccine (TIV) and most of the epidemic isolates at that time, and to discuss if there are some optimized programs for seasonal influenza vaccines. Methods: HA and NA sequences of the seasonal isolates circulating from December 1, 2017 to February 28, 2018, and in the previously other 7 winters in northern hemisphere from Global Initiative on Sharing All Influenza Data (GISAID) and the influenza database of National Center for Biotechnology Information (NCBI). Phylogenetic trees and genetic distances were constructed or calculated by using MAFFT and MEGA 6.0 software. Results: Influenza B composition in the TIV recommendation mismatched most of circulating viruses in 2017/2018 winter; the vaccine strain was from the B/Victoria lineage, while most of epidemic isolates were from the B/Yamagata lineage. The epidemic lineage of influenza B reached its peak a little late in the previous winter might be responsible for this mismatch. During 2010-2018, the mean genetic distances between epidemic isolates of influenza A (H1N1 and H3N2) and the vaccines were no higher than 0.02375 ± 0.00341 in both HA and NA. However, concerning influenza B virus, when forecasting done well, the mean genetic distances between epidemic isolates and the vaccines were no higher than 0.02368 ± 0.00272; otherwise, the distances could reach 0.13695 ± 0.00238. Conclusion: When applying quadrivalent influenza vaccines (QIVs) for vaccination, the recommendations of compositions for influenza B could be altered and assessed once in 3 or 4 years; when economic burden was considered intensively and TIVs were utilized, the recommended compositions for influenza B could be announced in April or May, rather than in February or March as now.