R229I substitution from oseltamivir induction in HA1 region significantly increased the fitness of a H7N9 virus bearing NA 292K.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

Construction and mechanistic insights of a novel ZnO functionalized rGO composite for efficient adsorption and reduction of Cr(VI).

A series of ZnO decorated reduced graphene oxide (rGO) (ZnrGOx) with different doping ratios were synthesized by the alkaline hydrothermal method using graphene oxide (GO) and Zn(NO3)2·6H2O as precursors, and subsequently used for the adsorption study of Cr(VI) in water. The morphology, crystalline phase structure, and surface elemental properties of ZnrGOx composites were revealed by XRD, SEM, BET, FT-IR, and XPS characterizations. The results showed that ZnO nanoparticles can be clearly seen on the surface of layered rGO. Meanwhile, as the doping rate increased, the C = C double bonds were broken and more carboxylic acid groups formed in ZnrGOx. In addition, the ZnrGO0.1 composite had the most excellent adsorption performance and good stability, and reusability. The adsorption removal rate of Cr(VI) can reach 99%, and the maximum adsorption amount of Cr(VI) was 68.9655 mg/g in 3 h. The isothermal and kinetic model simulations showed that Cr(VI) adsorption on ZnrGO0.1 composite is a chemical adsorption process, spontaneous and endothermic. Based on the concentrations of different valence states of Cr in the solid and liquid phases, 40% of Cr(VI) was reduced to Cr(III) on the surface of ZnrGO0.1 composite. Moreover, the adsorption-reduction mechanisms of Cr(VI) on ZnrGO0.1 composite were further elucidated. The ZnrGO0.1 composite manifested great potential as an efficient adsorbent for Cr(VI) removal.

Factors affecting the immunogenicity of influenza vaccines in human.

BACKGROUND: The influenza viruses pose a threat to human health and medical services, and vaccination is an important way to prevent infection. However, the effectiveness of influenza vaccines is affected by various aspects. This study aimed to explore factors related to the immune response to influenza vaccines. METHODS: The study was conducted from September 2019 to September 2021, and a total of 593 volunteers were recruited from the Center for Disease Control and Prevention in 3 provinces in China. The hemagglutination inhibition assay was used to measure antibody levels. The Chi-square test, multivariable logistic regression analysis, and sum-rank test were used to analyze the factors associated with influenza vaccine immune response. RESULTS: The Chi-square test showed that seroconversion rates and response rate were associated with age group, vaccination history, chronic conditions, the frequency of colds, and region (P < 0.05). The multivariable logistic regression analysis showed that age was an important factor that affected participants' seroconversion rates for A/H1N1, A/H3N2, B/Victoria, and response status (18-64 vs. ≤5: OR = 2.77, P < 0.001; ≥65 vs. ≤5: OR = 0.38, P = 0.01; 18-64 vs. ≤5: OR = 2.64, P = 0.03). Vaccination history was also an affecting factor for A/H1N1, B/Victoria, and response status (yes vs. no: OR = 0.4 / 0.44 / 0.25, P < 0.001). The frequency of colds and chronic conditions were also affecting factors for participants' seroconversion rates and response levels to different degrees. The sum-rank test showed that the fold changes for A/H1N1, B/Victoria, and B/Yamagata were associated with age group and vaccination history (P < 0.01). The fold changes for A/H3N2 were associated with the frequency of colds (P < 0.05), and those for B/Victoria were associated with gender and chronic conditions (P < 0.05). CONCLUSIONS: Vaccination history, age, health condition, and frequency of colds were important factors affecting the seroconversion rate of the influenza vaccine in human. There is a need for developing optimized vaccination strategies for vulnerable groups to improve the efficacy of influenza vaccines in human.

Genetic variants in BAT2 are associated with immune responsiveness to influenza vaccination.

Background: Influenza is a global public health problem for its detrimental impact on human health. Annual vaccination is the most effective prevention of influenza infection. Identifying host genetic factors associated with the responsiveness to influenza vaccines can provide clues for developing more effective influenza vaccines. In this study, we aimed to explore whether the single nucleotide polymorphisms in BAT2 are associated with the antibody responses to influenza vaccines. Method: A nested case-control study was conducted in this research. 1968 healthy volunteers were enrolled and 1,582 of them from a Chinese Han population were eligible for further research. According to the hemagglutination inhibition titers of subjects against all influenza vaccine strains, a total of 227 low responders and 365 responders were included in the analysis. Six tag single nucleotide polymorphisms in the coding region of BAT2 were selected and genotyped using the MassARRAY technology platform. Univariable and multivariable analyses were conducted to evaluate the relationship between variants and antibody responses to influenza vaccination. Results: Multivariable logistic regression analysis showed that, compared with the BAT2 rs1046089GG genotype, the GA + AA genotype was correlated with decreased risk of low responsiveness to influenza vaccines after adjusting for gender and age (p = 1.12E-03, OR = .562, 95%CI: .398-.795). rs9366785 GA + AA genotype was associated with a higher risk of low responsiveness to influenza vaccination compared with the GG genotype (p = .003, OR = 1.854, 95%CI: 1.229-2.799). The haplotype consisting of BAT2 rs2280801-rs10885-rs1046089-rs2736158-rs1046080-rs9366785 CCAGAG was correlated with a higher level of antibody response to influenza vaccines compared with haplotype CCGGAG (p < .001, OR = .37, 95%CI: .23-.58). Conclusion: Genetic variants in BAT2 were statistically associated with the immune response to influenza vaccination among the Chinese population. Identifying these variants will provide clues for further research on novel broad-spectrum influenza vaccines, and improve the individualized influenza vaccination scheme.

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.

Single Nucleotide Polymorphisms in the Human Leukocyte Antigen Region Are Associated With Hemagglutination Inhibition Antibody Response to Influenza Vaccine.

Background: The annual death associated with seasonal influenza is 290,000-650,000 globally, which can be effectively reduced by influenza vaccination. However, the protective hemagglutination inhibition (HAI) antibody response to influenza vaccine is affected by many factors, among which single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region can alter the antigen-presenting function of the HLA molecule, thus influencing the process of antibody mounting against vaccine antigen. Methods: Healthy subjects of the Han nationality were recruited and received seasonal trivalent influenza vaccine. Paired serum samples collected on and approximately 28 days after vaccination were tested in parallel by HAI assays. HLA alleles related to the immune response to influenza vaccine reported in the previous literature were summarized, and six corresponding tag SNPs were selected and genotyped using the MassARRAY technology platform. Results: The effects of HLA SNPs on HAI antibody response to influenza vaccine varied with different vaccine antigens. The AA genotype of rs41547618 was correlated with low A/H1N1-specific antibody titer compared with the GG + GA genotype (p = .007). The TT genotype of rs17885382 was correlated with low A/H3N2-specific antibody titer compared with the CC + CT genotype (p = .003). In addition, haplotype consisting of rs41542812-rs17885382-rs2068205-rs41547618-rs6905837-rs9270299-CCTGCA was correlated with non-responsiveness to influenza vaccine (OR = 2.39, 95% CI = 1.02-5.62). Conclusion: HLA SNPs were associated with HAI antibody response to influenza vaccine, which can help in a better understanding of the varied responsiveness to influenza vaccine in the population.

Association of Single Nucleotide Polymorphisms in LEP, LEPR, and PPARG With Humoral Immune Response to Influenza Vaccine.

Background: Although previous studies have proposed leptin plays an important role in energy metabolism as well as in immune response, the effects of leptin-related genes on influenza vaccine-induced immune response remain unexplored. In this study, we aimed to investigate the potential association of leptin gene (LEP), leptin receptor gene (LEPR), and peroxisome proliferator activated receptor gamma gene (PPARG) polymorphisms with humoral immune response to influenza vaccine. Methods: Based on the seroconversion to influenza vaccine, 227 low-responders and 365 responders were selected in this study, and 11 candidate single nucleotide polymorphisms (SNPs) were genotyped using the MassARRAY technology platform. Univariate and multivariate logistic regression analyses were used to explore the association of SNPs in LEP, LEPR, and PPARG with humoral immune response to influenza vaccine. We also conducted a stratified analysis by gender to further clarify this association. The haplotypes analysis was performed using SNPStats. Results: Significant differences were observed in the genotypic distribution of PPARG rs17793951 between the two groups (p = 0.001), and the PPARG rs17793951 AG + GG genotype was associated with a higher risk of low responsiveness to influenza vaccine adjusted for gender and age (additive genetic model: OR = 2.94, 95% CI = 1.67-5.19, dominant genetic model: OR = 2.81, 95% CI = 1.61-4.92). No significant association of other SNPs in LEP and LEPR with immune response to influenza vaccine was found. The stratified analysis found the gender difference in the association of LEPR and PPARG variants with immune response to influenza vaccine. We found that LEPR rs6673591 GA + AA genotype was correlated with low responsiveness to influenza vaccine only in males (OR = 1.96, 95% CI = 1.05-3.67), and PPARG rs17793951 AG + GG genotype was associated with low responsiveness to influenza vaccine in females (OR = 3.28, 95% CI = 1.61-6.67). Compared with the CGGAGGC haplotype composed of LEPR rs1327118, rs7602, rs1137101, rs1938489, rs6673591, rs1137100, and rs13306523, the CAAAAAC haplotype was positively correlated with immune response of influenza vaccine (OR = 0.34, 95% CI = 0.15-0.77). Haplotype TG comprised of PPARG rs796313 and rs17793951 was associated with a 2.85-fold increased risk of low responsiveness to influenza vaccine. Conclusion: Our study identified that PPARG rs17793951 variants were significantly associated with the immune response to influenza vaccine.

Epidemiological and Virological Surveillance of Seasonal Influenza Viruses - China, 2020-2021.

INTRODUCTION: During the coronavirus disease 2019 (COVID-19) pandemic, the circulation of seasonal influenza virus declined globally and remained below previous seasonal levels. We analyzed the results of the epidemiology, antigenic, and genetic characteristics, and antiviral susceptibilities of seasonal influenza viruses isolated from the mainland of China during October 5, 2020 through September 5, 2021, to better assess the risk of influenza during subsequent influenza season in 2021-2022. METHODS: Positive rates of influenza virus detection during this period were based on real-time polymerase chain reaction (PCR) detection by the Chinese National Influenza Surveillance Network laboratories, and isolated viruses from influenza positive samples were submitted to the Chinese National Influenza Center. Antigenic analyses for influenza viruses were conducted using the hemagglutination inhibition assay. Next-generation sequencing was used for genetic analyses. Viruses were tested for resistance to antiviral medications using a phenotypic assay and next-generation sequencing. RESULTS: In southern China, the influenza positivity rate was elevated especially after March 2021 and was higher than the same period the previous year with the COVID-19 pandemic. In northern China, influenza positive rate peaked at Week 18 in 2021 and has declined since then. Nearly all isolated viruses were B/Victoria lineage viruses during the study period, and 37.3% of these viruses are antigenically similar to the reference viruses representing the vaccine components for the 2020-2021 and 2021-2022 Northern Hemisphere influenza season. All seasonal influenza viruses were susceptible to neuraminidase inhibitors and endonuclease inhibitors. CONCLUSIONS: Influenza activity has gradually increased in the mainland of China in 2021, although the intensity of activity is still lower than before the COVID-19 pandemic. The diversity of circulating influenza types/subtypes decreased, with the vast majority being B/Victoria lineage viruses. The surveillance data from this study suggest that we should strengthen influenza surveillance during the upcoming traditional influenza season. It also provided evidence for vaccine recommendations and prevention and control of influenza and clinical use of antiviral drugs.

Identification of Two Novel Candidate Genetic Variants Associated With the Responsiveness to Influenza Vaccination.

Background: Annual vaccination is the most effective prevention of influenza infection. Up to now, a series of studies have demonstrated the role of genetic variants in regulating the antibody response to influenza vaccine. However, among the Chinese population, the relationship between genetic factors and the responsiveness to influenza vaccination has not been clarified through genome-wide association study (GWAS). Method: A total of 1,968 healthy volunteers of Chinese descent were recruited and 1,582 of them were available for the subsequent two-stage analysis. In the discovery stage, according to our inclusion criteria, 123 of 1,582 subjects were selected as group 1 and received whole-genome sequencing to identify potential variants and genes. In the verification stage, 29 candidate variants identified by GWAS were selected for further validation in 481 subjects in group 2. Besides, we also analyzed nine variants from previously published reports in our study. Results: Multivariate logistic regression analysis showed that compared with the TT genotype of ZBTB46 rs2281929, the TC + CC genotype was associated with a lower risk of low responsiveness to influenza vaccination adjusted for gender and age (Group 2: P = 7.75E-05, OR = 0.466, 95%CI = 0.319-0.680; Combined group: P = 1.18E-06, OR = 0.423, 95%CI = 0.299-0.599). In the combined group, IQGAP2 rs2455230 GC + CC genotype was correlated with a lower risk of low responsiveness to influenza vaccination compared with the GG genotype (P = 8.90E-04, OR = 0.535, 95%CI = 0.370-0.774), but the difference was not statistically significant in group 2 (P = 0.008). The antibody fold rises of subjects with ZBTB46 rs2281929 TT genotype against H1N1, H3N2,and B were all significantly lower than that of subjects with TC + CC genotype (P < 0.001). Compared with IQGAP2 rs2455230 GC + CC carriers, GG carriers had lower antibody fold rises to H1N1 (P = 0.001) and B (P = 0.032). The GG genotype of rs2455230 tended to be correlated with lower antibody fold rises (P = 0.096) against H3N2, but the difference was not statistically significant. No correlation was found between nine SNPs from previously published reports and the serological response to influenza vaccine in our study. Conclusion: Our study identified two novel candidate missense variants, ZBTB46 rs2281929 and IQGAP2 rs2455230, were associated with the immune response to influenza vaccination among the Chinese population. Identifying these variants will provide more evidence for future research and improve the individualized influenza vaccination program.

Incidence of influenza virus infections confirmed by serology in children and adult in a suburb community, northern China, 2018-2019 influenza season.

BACKGROUND: In mainland China, seasonal influenza disease burden at community level is unknown. The incidence rate of influenza virus infections in the community is difficult to determine due to the lack of well-defined catchment populations of influenza-like illness surveillance sentinel hospitals. OBJECTIVES: We established a community-based cohort to estimate incidence of seasonal influenza infections indicated by serology and protection conferred by antibody titers against influenza infections during 2018-2019 influenza season in northern China. METHODS: We recruited participants in November 2018 and conducted follow-up in May 2019 with collection of sera every survey. Seasonal influenza infections were indicated by a 4-fold or greater increase of hemagglutination inhibition (HI) antibody between paired sera. RESULTS: Two hundred and three children 5-17 years of age and 413 adults 18-59 years of age were followed up and provided paired sera. The overall incidence of seasonal influenza infection and incidence of A(H3N2) infection in children (31% and 17%, respectively) were significantly higher than those in adults (21% and 10%, respectively). The incidences of A(H1N1)pdm09 infection in children and adults were both about 10%, while the incidences of B/Victoria and/Yamagata infection in children and adults were from 2% to 4%. HI titers of 1:40 against A(H1N1)pdm09 and A(H3N2) viruses were associated with 63% and 75% protection against infections with the two subtypes, respectively. CONCLUSIONS: In the community, we identified considerable incidence of seasonal influenza infections. A HI titer of 1:40 could be sufficient to provide 50% protection against influenza A virus infections indicated by serology.

Exosomal Transfer of Macrophage-Derived miR-223 Confers Doxorubicin Resistance in Gastric Cancer.

PURPOSE: Macrophages are a major component of the tumour microenvironment and play an important role in chemoresistance of cancer. However, how exosomal microRNAs (miRNAs) derived from macrophages contribute to the development of doxorubicin resistance in gastric cancer (GC) are not clearly defined. The aim of this study was to investigate whether macrophage-derived exosomes mediate doxorubicin resistance in GC. METHODS: Exosomes isolated from macrophage culture medium were characterized and co-cultured with GC cells and the miR-223 level was detected using real-time quantitative PCR (RT-qPCR). The internalization of exosomes and transfer of miR-223 were observed via immunofluorescence. Macrophages were transfected with an miR-223 inhibitor or negative control. Cell Counting Kit-8 and flow cytometry were employed to explore the effect of macrophage-derived exosomes on the doxorubicin resistance of GC cells. Western blot and RT-qPCR assay were also performed to explore the regulation of GC chemotherapy resistance by exosomal miR-223. RESULTS: Here, the macrophages and macrophage-derived exosomes promoted doxorubicin resistance in GC cells. MiR-223 was enriched in macrophage-derived exosomes and they could be transferred to co-cultivated GC cells. The miR-223 knockdown in macrophages could reduce the effects of exosomes on GC cells. Functional studies revealed that exosomal miR-223 derived from macrophages promoted doxorubicin resistance in GC cells by inhibiting F-box and WD repeat domain-containing 7 (FBXW7). Clinically, the expression of miR-223 significantly increased in GC tissues and high expression of plasma exosomal miR-223 was highly linked with doxorubicin resistance in GC patients. CONCLUSION: The exosomal transfer of macrophage-derived miR-223 conferred doxorubicin resistance in GC and targeting exosome communication may be a promising new therapeutic strategy for GC patients.

Effectiveness of favipiravir (T-705) against wild-type and oseltamivir-resistant influenza B virus in mice.

The emergence of resistant mutants to the wildly used neuraminidase inhibitors (NAIs) makes the development of novel drugs necessary. Favipiravir (T-705) is one of the RNA-dependent RNA polymerase (RdRp) inhibitors developed in recent years. To examine the efficacy of T-705 against influenza B virus infections in vivo, C57BL/6 mice infected with wild-type or oseltamivir-resistant influenza B/Memphis/20/96 viruses were treated with T-705. Starting 2 h post inoculation (hpi), T-705 was orally administered to mice BID at dosages of 50, 150, or 300 mg/kg/day for 5 days. Oseltamivir was used as control. Here, we showed that T-705 protected mice from lethal infection in a dose-dependent manner. T-705 administration also significantly reduced viral loads and suppressed pulmonary pathology. In addition, phenotypic assays demonstrated that no T-705-resistant viruses emerged after T-705 treatment. In conclusion, T-705 can be effective to protect mice from lethal infection with both wild-type and oseltamivir-resistant influenza B viruses.

Human infection with a novel reassortant Eurasian-avian lineage swine H1N1 virus in northern China.

Influenza A virus infections occur in different species, causing mild to severe respiratory symptoms that lead to a heavy disease burden. Eurasian avian-like swine influenza A(H1N1) viruses (EAS-H1N1) are predominant in pigs and occasionally infect humans. An influenza A(H1N1) virus was isolated from a boy who was suffering from fever and headache and designated as A/Tianjin-baodi/1606/2018(H1N1). Full-genome sequencing and phylogenetic analysis revealed that A/Tianjin-baodi/1606/2018(H1N1) is a novel reassortant EAS-H1N1 containing gene segments from EAS-H1N1 (HA and NA), classical swine H1N1(NS) and A(H1N1)pdm09(PB2, PB2, PA, NP and M) viruses. The isolation and analysis of A/Tianjin-baodi/1606/2018(H1) provide further evidence that EAS-H1N1 poses a threat to human health and greater attention should be paid to surveillance of influenza virus infection in pigs and humans.

Highly pathogenic avian influenza H7N9 viruses with reduced susceptibility to neuraminidase inhibitors showed comparable replication capacity to their sensitive counterparts.

BACKGROUND: Human infection with avian influenza H7N9 virus was first reported in 2013. Since the fifth epidemic, a highly pathogenic avian influenza (HPAI) H7N9 virus has emerged and caused 33 human infections. Several potential NAI resistance sites have been found in human cases. However, the drug susceptibility and replication ability of HPAI H7N9 virus with such substitutions have not yet been studied. METHODS: Thirty-three HPAI H7N9 virus strains were isolated from human cases in China, and then sequences were analyzed to identify potential NAI resistance sites. Recombinant influenza viruses were generated to evaluate the effect of NA amino acid substitutions on NAI (oseltamivir or zanamivir) susceptibility and viral replication efficiency in MDCK cells. RESULTS: Four potential NAI resistance sites, R292 K, E119V, A246T or H274Y, were screened. All four substitutions conferred either reduced or highly reduced susceptibility to oseltamivir or zanamivir. 292 K not only highly reduced the susceptibility of HPAI H7N9 to oseltamivir but also induced an increase in the IC50 of zanamivir. 119 V or 274Y conferred reduced susceptibility of HPAI H7N9 to oseltamivir. Additionally, 246 T conferred reduced susceptibility to zanamivir. All tested NAI-resistant viruses were capable of replication in MDCK cells. The virus yields of rg006-NA292K were lower than those of rg006-NA292R at 24, 48, 72 and 96 h postinfection (P<0.05). Rg006-NA119V, rg006-NA246T or rg006-NA274Y showed comparable replication capacity to wild-type virus (except for rg006-NA274Y at 96 h, P<0.05). CONCLUSIONS: All 4 amino acid substitutions (R292 K, E119V, A246T or H274Y) in NA reduced the susceptibility of HPAI H7N9 to NAIs. The NAI-resistant mutations in HPAI H7N9, in most cases, did not reduce the replication ability of the virus in mammalian cells. Special attention needs to be paid to these mutations, and the development of new anti-H7N9 drugs is of great importance.

Neuraminidase inhibitor susceptibility profile of human influenza viruses during the 2016-2017 influenza season in Mainland China.

To understand the current situation of antiviral-resistance of influenza viruses to neuraminidase inhibitors (NAIs) in Mainland China, The antiviral-resistant surveillance data of the circulating influenza viruses in Mainland China during the 2016-2017 influenza season were analyzed. The total 3215 influenza viruses were studied to determine 50% inhibitory concentration (IC50) for oseltamivir and zanamivir using a fluorescence-based assay. Approximately 0.3% (n = 10) of viruses showed either highly reduced inhibition (HRI) or reduced inhibition (RI) against at least one NAI. The most common neuraminidase (NA) amino acid substitution was H275Y in A (H1N1)pdm09 virus, which confers HRI by oseltamivir. Two A (H1N1)pdm09 viruses contained a new NA amino acid substitution respectively, S110F and D151E, which confers RI by oseltamivir or/and zanamivir. Two B/Victoria-lineage viruses harbored a new NA amino acid substitution respectively, H134Q and S246P, which confers RI by zanamivir. One B/Victoria-lineage virus contained dual amino acid substitution NA P124T and V422I, which confers HRI by zanamivir. One B/Yamagata-lineage virus was a reassortant virus that haemagglutinin (HA) from B/Yamagata-lineage virus and NA from B/Victoria-lineage virus, defined as B/Yamagata-lineage virus confers RI by oseltamivir, but as B/Victoria-lineage virus confers normal inhibition by oseltamivir. All new substitutions that have not been reported before, the correlation of these substitutions and observed changes in IC50 should be further assessed. During the 2016-2017 influenza season in Mainland China the majority tested viruses were susceptible to oseltamivir and zanamivir. Hence, NAIs remain the recommended antiviral for treatment and prophylaxis of influenza virus infections.

Effect of Oseltamivir on the Hemagglutination Test and Hemagglutination Inhibition Test of the Influenza A(H3N2) Virus in China.

We compared the effect of oseltamivir on the hemagglutination (HA) test and hemagglutinin inhibition (HI) test of the influenza A(H3N2) virus in China to obtain the "true" HA titer and antigenic variation. A total of 395 influenza H3N2 strains isolated in mainland China from October 2014 to May 2015 were analyzed with HA and HI tests, with or without oseltamivir.. Gene sequencing was undertaken for selected viruses, and the amino-acid sequence of neuraminidase (NA) protein was compared with the vaccine strain. In the HA test in the presence of oseltamivir, the HA titer was unchanged in 44. 8%, decreased in 43. 8%, and increased in 11. 4% of tested strains. In the presence of oseltamivir, the proportion of viruses similar to A/TX/50/2012 egg isolates was significantly higher, and the proportion of viruses similar to A/SZ/9715293/2013 cell isolates significantly lower, than the proportion obtained from the test without the presende of oseltamivir. A significant difference was detected between the tests with or without oseltamivir. In A/TX/50/2012 cell isolates and A/SZ/9715293/2013 egg isolates, no significant difference was detected between the tests with or without oseltamivir. Nineteen selected strains' of influenza A ( H3N2) were sequenced, and the amino-acid sites were compared with A/TX/50/2012 egg isolates. Five strains had a more-than-fourfold decrease in HA titer when addition of oseliamivir showed no common mutation in amino acids, whereas the A/Shandong Laicheng/119/2015 strain had a D151G mutation and the A/Jilin Tiexi/1194/2015 strain had a V4121 and T434A mutation in the NA protein. The strain had a two-to-fourfold decrease in HA titer when addition of oseltamivir showed 126T, G93S, V1491, N234D, T267K and S416G mutations in NA protein.. These data show that, for recent circulating influenza H3N2 viruses, the presence of oseltamivir can be used to obtain more accurate HA and HI titers for antigenic analysis and vaccine evaluations.

[Susceptibility of human influenza A (H3N2) viruses to neuraminidase inhibitors isolated during 2011-2012 in China].

OBJECTIVE: To analyze the susceptibility of influenza A (H3N2) viruses to neuraminidase inhibitors during 2011-2012 in Mainland China. METHODS: All the tested viruses were obtained from the Chinese National Influenza Surveillance Network, which covers 31 provinces in mainland China, including 408 network laboratories and 554 sentinel hospitals. In total 1 903 viruses were selected with isolation date from January 1, 2011 to December 31, 2012 in Mainland China, among these viruses, 721 were confirmed to be influenza A (H3N2) virus by Chinese National Influenza Center and tested for the susceptibility to oseltamivir and zanamivir using chemiluminescence-based assay. The neuraminidase inhibitor sensitive reference virus A/Washington/01/2007 (119E) and oseltamivir resistant virus A/Texas/12/2007 (E119V) were used as control in this study. The t -test was used to compare the difference of NAI susceptibility of viruses isolated from different years. RESULTS: The half maximal inhibitory concentration (IC50) of A/Washington/01/2007 for oseltamivir and zanamivir was (0.10 ± 0.02) and (0.30 ± 0.05) nmol/L, respectively. The IC50 of A/Texas/12/2007 for oseltamivir and zanamivir was (4.27 ± 1.60) and (0.20 ± 0.03) nmol/L, respectively. Among the 721 influenza A (H3N2) viruses, 132 influenza A (H3N2) viruses were isolated in 2011 and 589 influenza A (H3N2) viruses were isolated in 2012. The IC50 for oseltamivir ranged from 0.04 to 0.62 nmol/L for viruses isolated in 2011 and ranged from 0.02 to 0.95 nmol/L for viruses in 2012, and the IC50 of all the viruses tested was within 10-fold IC50 (1.0 nmol/L) of the neuraminidase inhibitor sensitive reference virus A/Washington/01/2007. The IC50 of zanamivir ranged from 0.12 to 0.80 nmol/L for viruses in 2011 and ranged from 0.04 to 0.72 nmol/L for viruses in 2012, and was within 10-fold IC50 (3.0 nmol/L) of the neuraminidase inhibitor sensitive reference virus A/Washington/01/2007. CONCLUSION: The influenza A(H3N2) viruses isolated during 2011-2012 in Mainland China were tested to be sensitive to oseltamivir and zanamivir.

[Susceptibility of Influenza B Viruses to Neuraminidase Inhibitors Isolated during 2013-2014 Influenza Season in Mainland China].

Data based on the antiviral-resistant phenotyping characteristics of 884 influenza B viruses circulating in mainland China from October 2013 to March 2014 were analyzed to assess the susceptibility of influenza B viruses to neuraminidase inhibitors. All 884 viruses were sensitive to oseltamivir; two viruses (0.23%) had reduced sensitivity to zanamivir and all other viruses were sensitive to zanamivir. Among the 38 viruses with a B/Victoria lineage, B/Shandong-Kuiwen/1195/2014 exhibited a half-maximal inhibitory concentration (IC50) for zanamivir that was elevated by 5. 12-fold (1.78 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D35G, N59D and S402T (39, 64 and 399 with N2 number) amino-acid substitutions in the NA gene were detected with no previously reported antiviral-resistant substitutions. Among viruses with the 846 B/Yamagata lineage, B/Hunan-Lingling/350/2013 exhibited a 7.99-fold elevated IC50 for zanamivir (2.72 nM) compared with neuraminidase inhibitors sensitive to the reference virus (0.34 nM), suggesting that it exhibited reduced inhibition by zanamivir. D197N (N2 number), a previously reported antiviral resistant-related amino-acid substitution in the NA gene, was detected in B/Hunan-Lingling/350/2013. These data suggest that recently circulating influenza B viruses in mainland China have retained susceptibility to neuraminidase inhibitors.