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.

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.

An Inexpensive and Accurate Reverse Transcription-PCR-Melting Temperature Analysis Assay for Real-Time Influenza Virus B Lineage Discrimination.

In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm ) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010-2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r = 0.287, P = 2.245 × 10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.

Co-circulation of the two influenza B lineages during 13 consecutive influenza surveillance seasons in Italy, 2004-2017.

BACKGROUND: Since 1985, two antigenically distinct lineages of influenza B viruses (Victoria-like and Yamagata-like) have circulated globally. Trivalent seasonal influenza vaccines contain two circulating influenza A strains but a single B strain and thus provide limited immunity against circulating B strains of the lineage not included in the vaccine. In this study, we describe the characteristics of influenza B viruses that caused respiratory illness in the population in Italy over 13 consecutive seasons of virological surveillance, and the match between the predominant influenza B lineage and the vaccine B lineage, in each season. METHODS: From 2004 to 2017, 26,886 laboratory-confirmed influenza cases were registered in Italy, of which 18.7% were type B. Among them, the lineage of 2465 strains (49%) was retrieved or characterized in this study by a real-time RT-PCR assay and/or sequencing of the hemagglutinin (HA) gene. RESULTS: Co-circulation of both B lineages was observed each season, although in different proportions every year. Overall, viruses of B/Victoria and B/Yamagata lineages caused 53.3 and 46.7% of influenza B infections, respectively. A higher proportion of infections with both lineages was detected in children, and there was a declining frequency of B/Victoria detections with age. A mismatch between the vaccine and the predominant influenza B lineage occurred in eight out of thirteen influenza seasons under study. Considering the seasons when B accounted for > 20% of all laboratory-confirmed influenza cases, a mismatch was observed in four out of six seasons. Phylogenetic analysis of the HA1 domain confirmed the co-circulation of both lineages and revealed a mixed circulation of distinct evolutionary viral variants, with different levels of match to the vaccine strains. CONCLUSIONS: This study contributes to the understanding of the circulation of influenza B viruses in Italy. We found a continuous co-circulation of both B lineages in the period 2004-2017, and determined that children were particularly vulnerable to Victoria-lineage influenza B virus infections. An influenza B lineage mismatch with the trivalent vaccine occurred in about two-thirds of cases.

Development of high-yield influenza B virus vaccine viruses.

The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates. Therefore, we screened virus libraries possessing random mutations in the six "internal" influenza B viral RNA segments [i.e., those not encoding the major viral antigens, hemagglutinin (HA) and neuraminidase NA)] for mutants that confer efficient replication. Candidate viruses that supported high yield in cell culture were tested with the HA and NA genes of eight different viruses of the Victoria and Yamagata lineages. We identified combinations of mutations that increased the titers of candidate vaccine viruses in mammalian cells used for human influenza vaccine virus propagation and in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus vaccine backbones can be used for improved vaccine virus production.

Genetic characterization of influenza B virus in Cameroon and high frequency of reassortant strains.

Influenza B is broadly divided into B/Victoria and B/Yamagata lineages based on its genetic and antigenic properties. We describe in this study the first report on genome characterization of type B influenza virus in the Cameroon National Influenza Center (NIC) between 2014 and 2017. Respiratory samples were collected as part of the influenza surveillance activity in the NIC. RNA products were tested for the presence of influenza using the CDC Influenza A/B typing panel. Thirty-five samples positive for influenza B were selected for sequencing three gene segments (HA, NA, and M) and phylogenetic trees were generated by MEGA version 6.0. Nucleotide phylogenetic analysis of the HA gene revealed the presence of three major clades among Cameroonian strains. All Victoria lineages grouped into B/Victoria clade 1A, while, Yamagata lineages grouped into Yamagata clade 2 (2014 strains) and Yamagata clade 3 (2015-2017). We observed a high frequency of reassortant viruses with Yamagata-like HA gene and Victoria-like NA gene (27.4%; 23/84). The results from this study confirm variations in the genome composition of type B influenza virus and emphasize on the relevance of molecular surveillance for spotting peculiar genetic variants of public health and clinical significance.

Comparative Epidemiology of Influenza B Yamagata- and Victoria-Lineage Viruses in Households.

Influenza B viruses split into 2 distinct lineages in the early 1980s, commonly named the Victoria and Yamagata lineages. There are few data on the comparative epidemiology of Victoria- and Yamagata-lineage viruses. In 2007-2011, we enrolled 75 and 34 households containing index patients with acute respiratory illness who tested positive for Yamagata- and Victoria-lineage viruses, respectively, from outpatient clinics in Hong Kong, China. These index patients and their household contacts were followed up for 7-10 days. We examined overall risk of polymerase chain reaction-confirmed infection among household contacts and the risk of secondary infection within households using an individual-based hazard model that accounted for tertiary transmission and infections occurring outside the household. We found that for Victoria-lineage viruses, the risk of within-household infection among household contacts aged ≤15 years was significantly higher (risk ratio = 12.9, 95% credibility interval: 4.2, 43.6) than that for older household contacts, while for Yamagata-lineage viruses, the risk of within-household infection for household contacts did not differ by age. Influenza B Yamagata- and Victoria-lineage viruses have similar characteristics in terms of viral shedding and clinical illness. The mechanisms underlying these epidemiologic differences deserve further investigation.

Multivalent Epigraph Hemagglutinin Vaccine Protects against Influenza B Virus in Mice.

Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only induce strain-specific responses and have suboptimal efficacy when mismatched from circulating strains. Further, two influenza B virus lineages have been described, B/Yamagata-like and B/Victoria-like, and the limited cross-reactivity between the two lineages provides an additional barrier in developing a universal influenza B virus vaccine. Here, we report a novel multivalent vaccine using computationally designed Epigraph hemagglutinin proteins targeting both the B/Yamagata-like and B/Victoria-like lineages. When compared to the quadrivalent commercial vaccine, the Epigraph vaccine demonstrated increased breadth of neutralizing antibody and T cell responses. After lethal heterologous influenza B virus challenge, mice immunized with the Epigraph vaccine were completely protected against both weight loss and mortality. The superior cross-reactive immunity conferred by the Epigraph vaccine immunogens supports their continued investigation as a universal influenza B virus vaccine.

In-field detection and characterization of B/Victoria lineage deletion variant viruses causing early influenza activity and an outbreak in Louisiana, 2019.

Background: In 2019, the Louisiana Department of Health reported an early influenza B/Victoria (B/VIC) virus outbreak. Method: As it was an atypically large outbreak, we deployed to Louisiana to investigate it using genomics and a triplex real-time RT-PCR assay to detect three antigenically distinct B/VIC lineage variant viruses. Results: The investigation indicated that B/VIC V1A.3 subclade, containing a three amino acid deletion in the hemagglutinin and known to be antigenically distinct to the B/Colorado/06/2017 vaccine virus, was the most prevalent circulating virus within the specimens evaluated (86/88 in real-time RT-PCR). Conclusion: This work underscores the value of portable platforms for rapid, onsite pathogen characterization.

Evolutionary pattern of reemerging influenza B/Victoria lineage viruses in São Paulo, Brazil, 1996-2012: Implications for vaccine composition strategy.

Since the 1980s, 2 antigenically distinct influenza B lineages have cocirculated in the world: B/Victoria/2/87 (first appeared in the 1980s) and B/Yamagata/16/88 (became predominant in the 1990s). B/Victoria/2/87 isolates were geographically restricted to eastern Asia during 1991-2000. During 2000-2001 and 2001-2002, B/Victoria/2/87 isolates reemerged in North America, Europe, and South America, and then spread globally. During influenza virus surveillance, season 2002, an outbreak of acute respiratory illness, which quickly spread among the population, has been notified by public health authorities living in Araraquara, São Paulo, Brazil. Instituto Adolfo Lutz and Secretariat of Health of São Paulo state teams initiate an investigation towards to describe the pattern of infection in this population temporally and by age and to characterize the strains by virus isolation and hemagglutination inhibition assay. The outbreak lasted approximately 10 weeks; many cases occurred between mid-August and mid-September. Children younger than 13 years were the most affected; the elderly were mostly immune to infection. Analysis of the clinical respiratory samples helped in identifying the B/Hong Kong/330/2001 and B/Brisbane/32/2002 subtypes-recent variants of B/Victoria/02/88, a lineage restricted to Southeast Asia until 2001. The Araraquara outbreak confirms the reemergence of the B/Victoria viruses in South America and highlights the importance of monitoring local circulating strains, especially in light of the absence of cross-protection between antigenically distinct influenza lineages. Based on influenza virus surveillance, public health authorities worldwide should decide whether trivalent vaccines or quadrivalent vaccines (containing both influenza virus B lineages) are to be used in each country.

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.

Epidemiological and virological surveillance of influenza viruses in China during 2020-2021.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, seasonal influenza activity declined globally and remained below previous seasonal levels, but intensified in China since 2021. Preventive measures to COVID-19 accompanied by different epidemic characteristics of influenza in different regions of the world. To better respond to influenza outbreaks under the COVID-19 pandemic, we analyzed the epidemiology, antigenic and genetic characteristics, and antiviral susceptibility of influenza viruses in the mainland of China during 2020-2021. METHODS: Respiratory specimens from influenza like illness cases were collected by sentinel hospitals and sent to network laboratories in Chinese National Influenza Surveillance Network. Antigenic mutation analysis of influenza virus isolates was performed by hemagglutination inhibition assay. Next-generation sequencing was used for genetic analyses. We also conducted molecular characterization and phylogenetic analysis of circulating influenza viruses. Viruses were tested for resistance to antiviral medications using phenotypic and/or sequence-based methods. RESULTS: In the mainland of China, influenza activity recovered in 2021 compared with that in 2020 and intensified during the traditional influenza winter season, but it did not exceed the peak in previous years. Almost all viruses isolated during the study period were of the B/Victoria lineage and were characterized by genetic diversity, with the subgroup 1A.3a.2 viruses currently predominated. 37.8% viruses tested were antigenically similar to reference viruses representing the components of the vaccine for the 2020-2021 and 2021-2022 Northern Hemisphere influenza seasons. In addition, China has a unique subgroup of 1A.3a.1 viruses. All viruses tested were sensitive to neuraminidase inhibitors and endonuclease inhibitors, except two B/Victoria lineage viruses identified to have reduced sensitivity to neuraminidase inhibitors. CONCLUSIONS: Influenza activity increased in the mainland of China in 2021, and caused flu season in the winter of 2021-2022. Although the diversity of influenza (sub)type decreases, B/Victoria lineage viruses show increased genetic and antigenic diversity. The world needs to be fully prepared for the co-epidemic of influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus globally.

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

The influenza virus B belongs to the family Orthomyxoviriridae and to the genus Influenzavirus B. It has a negative RNA-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 genetic lineage; but in this year two antigenically and genetically distinct lineages emerged which were named B/Victoria/2/1987 and 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 acquires genes from the Yamagata lineage. It has been proposed that the differences in the evolutionary dynamics of the two lineages are due to the different binding preferences of influenza hemagglutinin to the cellular receptor. The Victoria lineage has shown the ability to bind to cell receptors with sialic acid residues at the α-2,3 and α-2,6 positions; whereas the Yamagata lineage does so exclusively in the human α-2,6 positions of the respiratory tract. Low circulation in recent months may have contributed to the temporary elimination ("extinction") of the Yamagata lineage. Since 2017, almost all of the strains of this lineage belong to clade 3A, when previously multiple circulating clades were detected. Although this clade 3A is diverse at the genetic level and has acquired surrogate mutations in the hemagglutinin gene, these have not determined significant antigenic changes that have made it necessary to replace its antigenic component (B/Pukhet/3073/2013) in the influenza vaccine since 2015.

[Phylogenetic and pathogenicity analysis of influenza B virus strain B/Guangxi-Jiangzhou/1352/2018].

Influenza B virus (IBV) is more likely to cause complications than influenza A virus (IAV) and even causes higher disease burden than IAV in a certain season, but IBV has received less attention. In order to analyze the genetic evolution characteristics of the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018), we constructed genetic evolution trees and analyzed the homology and different amino acids of hemagglutinin and neuraminidase referring to the vaccine strains recommended by World Health Organization (WHO). We found that strain B/Guangxi-Jiangzhou/1352/2018 was free of interlineage reassortment and poorly matched with the vaccine strain B/Colorado/06/2017 of the same year. We also determined the median lethal dose (LD50) and the pathogenicity of strain B/Guangxi-Jiangzhou/1352/2018 in mice. The results showed that the LD50 was 105.9 TCID50 (median tissue culture infective dose), the IBV titer in the lungs reached peak 1 d post infection and the mRNA level of the most of inflammatory cytokines in the lungs reached peak 12 h post infection. The alveoli in the lungs were severely damaged and a large number of inflammatory cells were infiltrated post infection. The study demonstrated that the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018) could infect mice and induce typical lung inflammation. This will facilitate the research on the pathogenesis and transmission mechanism of IBV, and provide an ideal animal model for evaluation of new vaccines, antiviral and anti-inflammatory drug.

Comparison of the Hemagglutination Inhibition Titers against Influenza Vaccine Strains in Japan from the 2017/2018 to 2021/2022 Seasons Using a Single Set of Serum Samples.

In Japan, inactivated influenza vaccines are used. We measured titers of antibodies to vaccine strains of three influenza types-influenza A (H1N1), influenza A (H3N2), and influenza B/Victoria-from the 2017/2018 to 2021/2022 seasons, but not for influenza A (H3N2) from the 2018/2019 season, using a single set of serum samples from 34 healthy volunteers, and assessed the consistency in antibody positivity between seasons. The antibody titers in the 2017/2018 season were used as a reference. The influenza A (H1N1) antibody titer in 2019/2020 did not differ significantly from that in the 2017/2018 season, but the titers varied in the two subsequent seasons. The influenza A (H3N2) antibody titers toward the 2019/2020, 2020/2021, and 2021/2022 seasonal viruses differed significantly from that in the 2017/2018 season. The influenza B/Victoria antibody titer toward the 2019/2020 seasonal antigen differed from that in the 2017/2018 season, and the antibody positivity was inconsistent between seasons; however, the antibody titer in the 2020/2021 season did not differ significantly from those in the prior two seasons, and the antibody positivity was consistent between seasons. Antibody titers and their consistency can be used to evaluate cross-immunity of antibodies.

Duration of fever and symptoms in influenza-infected children treated with baloxavir marboxil during the 2019-2020 season in Japan and detection of influenza virus with the PA E23K substitution.

Data on the clinical effectiveness of the novel anti-influenza drug baloxavir marboxil (baloxavir) in children remain limited. We conducted an observational study to compare the duration of fever and symptoms between baloxavir- and oseltamivir-treated children infected with influenza A and B. In total, 159 outpatients with influenza A(H1N1)pdm09 or B/Victoria-lineage infections, aged <19 years, during the 2019-2020 influenza season in Japan were enrolled and assessed the duration of fever and symptoms using the Kaplan-Meier method and a multivariate Cox proportional hazard regression model. Polymerase acidic (PA) variants were examined before and after baloxavir treatment. In the multivariable analysis, the duration of fever and symptoms was unaltered between the A(H1N1)pdm09 (n = 116) and B/Victoria-lineage (n = 43) groups. Conversely, the fever duration was marginally longer in the oseltamivir-treated group (n = 59) than in the baloxavir group (n = 100) (hazard ratio (HR) = 0.67, p = 0.05); however, the duration of symptoms was unaltered between the two groups (HR = 0.74, p = 0.11). No patient presented PA reduced susceptibility marker(s) before baloxavir treatment in the analyzed groups. The PA/E23K variant was detected in one case (1.5%, 1/66) of A(H1N1)pdm09 after baloxavir treatment. One case (2.0%, 1/50) of A(H1N1)pdm09 with an N295S substitution in neuraminidase was detected following oseltamivir treatment. These results suggested that the duration of fever was likely to be shorter with baloxavir than with oseltamivir, but the difference between influenza A (H1N1)pdm09 and B/Victoria-lineage was unclear. It is important to continue evaluating the clinical effectiveness of baloxavir and monitoring its drug susceptibility to the influenza virus.

Detection of Victoria lineage influenza B viruses with K162 and N163 deletions in the hemagglutinin gene, South Africa, 2018.

BACKGROUND: A group of Victoria lineage influenza B viruses with a two amino acid deletion in the hemagglutinin (HA) at residues K162 and N163, was detected during the 2016 to 2017 Northern Hemisphere influenza season and continues to spread geographically. We describe the first identification of viruses with these deletions from South Africa in 2018. METHODS: Nasopharyngeal samples were obtained from the syndromic surveillance programs. Real-time reverse transcription-polymerase chain reaction was used for virus detection and lineage determination. Influenza genetic characterization was done using next-generation sequencing on the MiSeq platform. The duration of virus circulation was determined using thresholds calculated using the Moving Epidemic Method; duration was used as an indicator of disease transmissibility and impact. RESULTS: In 2018, 42% (426/1015) of influenza-positive specimens were influenza B viruses. Of 426 influenza B-positive samples, 376 (88%) had the lineage determined of which 75% (283/376) were Victoria lineage. The transmissibility of the 2018 South African influenza season was high for a few weeks, although the severity remained moderate through most of the season. The sequenced 2018 South African Victoria lineage influenza B viruses clustered in sub-clade V1A.1 with the 162-163 deletions. CONCLUSIONS: We report the first detection of the 162-163 deletion variant of influenza B/Victoria viruses from South Africa in 2018, and suggest that this deletion variant replaced the previous circulating influenza B/Victoria viruses. These deletions putatively affect the antigenic properties of the viruses because they border an immune-dominant region at the tip of the HA. Therefore, close monitoring of these newly emerging viruses is essential.

Influenza B viruses from different genetic backgrounds are variably impaired by neuraminidase inhibitor resistance-associated substitutions.

Identifying evolutionary routes to antiviral resistance among influenza viruses informs molecular-based resistance surveillance and clinical decisions. To improve antiviral management and understand whether clinically identified neuraminidase (NA) inhibitor (NAI) resistance-associated markers affect influenza B viruses of the Victoria- or Yamagata-lineages differentially, we generated a panel of NAI-resistant viruses (carrying E105K, G145E, R150K, D197N, I221 L/N/T/V, H273Y, N294S, or G407S substitutions; B numbering) in B/Brisbane/60/2008 (BR/08) and B/Phuket/3073/2013 (PH/13). In both backgrounds, I221 L/N/T/V resulted in reduced or highly reduced inhibition (HRI) by one to three currently available NAIs. D197N reduced inhibition by all NAIs in BR/08 but only by oseltamivir and peramivir in PH/13; R150K caused HRI by all NAIs in PH/13. Although PH/13 generally retained or enhanced NA activity in the presence of the substitutions, enzymatic activity in BR/08 was detrimentally affected. Similarly, substrate affinity and catalysis were relatively stable in PH/13, but not in the BR/08 variants. E105K, R150K, and D197N attenuated replication efficiency of BR/08 in vitro and in mice; only E105K had this effect in PH/13. Notably, the I221 L/N/T/V substitutions did not severely impair replication, particularly in PH/13. Overall, our data show differential effects of NA substitutions in representative Victoria- and Yamagata-lineage viruses, suggesting distinct evolution of these viruses caused variable fitness and NAI susceptibility profiles when similar key NA substitutions arise. Because the viruses harboring the I221 NA substitutions displayed undiminished fitness and are commonly reported, this position is likely to be the most clinically relevant marker for NAI resistance among contemporary influenza B viruses.

Characterization of Influenza A and B Viruses Circulating in Southern China During the 2017-2018 Season.

The trivalent seasonal influenza vaccine was the only approved and available vaccine during the 2016-2018 influenza seasons. It did not include the B/Yamagata strain. In this study, we report an acute respiratory disease outbreak associated with influenza B/Yamagata infections in Guangzhou, Southern China (January through March, 2018). Among the 9914 patients, 2241 (22.6%) were positive for the influenza B virus, with only 312 (3.1%) positive for the influenza A virus. The influenza B/Yamagata lineage dominated during this period in Southern China. The highest incidence of influenza A virus infection occurred in the children aged 5-14 years. In contrast, populations across all age groups were susceptible to the influenza B virus. Phylogenetic, mutations, and 3D structure analyses of hemagglutinin (HA) genes were performed to assess the vaccine-virus relatedness. The recommended A/H1N1 vaccine strain (A/Michigan/45/2015) during both 2017-2018 and 2018-2019 was antigen-specific for these circulating isolates (clade 6B.1) in Spring 2018. An outbreak of influenza B/Yamagata (clade 3) infections in 2018 occurred during the absence of the corresponding vaccine during 2016-2018. The recommended influenza B/Yamagata vaccine strain (B/Phuket/3073/2013) for the following season (2018-2019) was antigen-specific. Although there were only a few influenza B/Victoria infections in Spring 2018, five amino acid mutations were identified in the HA antigenic sites of the 19 B/Victoria isolates (clade 1A), when compared with the 2016-2018 B/Victoria vaccine strain. The number was larger than expected and suggested that the influenza B HA gene may be more variable than previously thought. One of the mutations (K180N) was noted to likely alter the epitope and to potentially affect the viral antigenicity. Seven mutations were also identified in the HA antigenic sites of 2018-2020 B/Victoria vaccine strain, of which some or all may reduce immunogenicity and the protective efficacy of the vaccine, perhaps leading to more outbreaks in subsequent seasons. The combined epidemiological, phylogenetic, mutations, and 3D structural analyses of the HA genes of influenza strains reported here contribute to the understanding and evaluation of how HA mutations affect vaccine efficacy, as well as to providing important data for screening and selecting more specific, appropriate, and effective influenza vaccine candidate strains.

Immunogenicity and safety of an egg-based inactivated quadrivalent influenza vaccine (GC3110A) versus two inactivated trivalent influenza vaccines with alternate B strains: A phase â ¢ randomized clinical trial in adults.

Two antigenically distinct influenza B lineage viruses (Yamagata/Victoria) have been co-circulating globally since the mid-1980s. The quadrivalent influenza vaccine (QIV) may provide better protection against unpredictable B strains. We conducted a randomized, double-blind, phase III trial to evaluate the immunogenicity and safety of an egg-based inactivated, split-virion QIV (GC3110A). Subjects aged ≥ 19 years were randomized 2:1:1 to be vaccinated with QIV- GC3110A, trivalent influenza vaccine (TIV) containing the Yamagata lineage strain (TIV-Yamagata), or TIV containing the Victoria lineage strain (TIV-Victoria). Hemagglutination inhibition assays were performed 21 days post-vaccination. Solicited/unsolicited adverse events (AEs) were assessed within 21 days after vaccination, while serious AEs were reported up to six months after vaccination. A total of 1,299 were randomized to receive QIV-GC3110A (648 subjects), TIV-Yamagata (325 subjects), or TIV-Victoria (326 subjects). Compared to the TIVs, the QIV-GC3110A met the non-inferiority criteria for all four subtype/lineage strains with respect to the geometric mean titer (GMT) ratio and the difference of seroconversion rate. The safety profiles of QIV-GC3110A were consistent with those of TIV. In conclusion, QIV-GC3110A is safe, immunogenic, and comparable to strain-matched TIV.