Biological nitrogen fixation in the long-term nitrogen-fertilized and unfertilized paddy fields, with special reference to diazotrophic iron-reducing bacteria.

Biological nitrogen fixation (BNF) is important to sustain nitrogen fertility of paddy soil and rice yield, while could be affected by nitrogen fertilization. Iron-reducing bacteria, Anaeromyxobacter and Geobacter, are newly found diazotrophic bacteria predominant in paddy soil. Experimental field of this study is a long-term (35 years) nitrogen fertilized (6.0 g N/m2/year) and unfertilized paddy field, where ca. 70% of rice yield was obtained yearly in nitrogen unfertilized plot (443 ± 37 g/m2) compared to fertilized plot (642 ± 64 g/m2). Effects of long-term nitrogen fertilization/unfertilization on soil properties related to BNF were investigated with special reference to diazotrophic iron-reducing bacteria. Soil chemical/biochemical properties, soil nitrogen-fixing activity, and community composition of diazotrophic bacteria were similar between nitrogen fertilized and unfertilized plot soils. In both plot soils, Anaeromyxobacter and Geobacter were the most predominant diazotrophs. Their nifD transcripts were detected at similar level, while those of other general diazotrophs were under detection limit. It was concluded that long-term use/unuse of nitrogen fertilizer in this field did not affect the predominance and nitrogen-fixing activity of diazotrophic iron-reducing bacteria, composition of other general diazotrophs, and the resulting soil nitrogen-fixing activity. BNF, primarily driven by diazotrophic iron-reducing bacteria, might significantly contribute to sustain soil nitrogen fertility and rice yield in both plot soils. Appropriate soil management to maintain BNF, including diazotrophic iron-reducing bacteria, will be important for sustainable soil nitrogen fertility and rice production.

CtBP2 confers protection against oxidative stress through interactions with NRF1 and NRF2.

While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically.

Programmed expression of pro-apoptotic BMCC1 during apoptosis, triggered by DNA damage in neuroblastoma cells.

BACKGROUND: The multi-functional BMCC1 (BCH motif-containing molecule at the carboxyl terminal region 1)/PRUNE2 plays a clear role in suppression of tumor activity. In the patients with neuroblastoma (NB), reduced expression of BMCC1 in primary tumor tissues was associated with poor prognosis. By contrast, enforced expression of BMCC1 as well as elevated expression of BMCC1 in response to DNA-damage promotes apoptosis by abrogating Akt-mediated survival pathways. METHODS: We addressed molecular mechanisms underlying changes in regulation of BMCC1 expression during the process of apoptosis, which was promoted by a DNA-damaging drug Cisplatin (CDDP), in NB-derived cells. RESULTS: Elevated expression of BMCC1 was identified as an early response to DNA damage, which is accompanied by phosphorylation of ataxia telangiectasia mutated kinase (ATM) and accumulation of E2F1. Indeed, inhibition of ATM using an ATM inhibitor resulted in a decrease in expression of BMCC1 at mRNA levels. In addition, an E2F-binding sight was required for activation of BMCC1 promoter in response to DNA damage. On the other hand, knockdown of E2F1 yielded abrogated induction of BMCC1 in the cells after treatment with CDDP, suggesting that BMCC1 accumulation was caused by ATM-E2F1-dependent transcription. Finally, we demonstrated that full-length BMCC1 was proteolytically cleaved by apoptosis-activated caspase-9 during advanced stages of apoptosis in SK-N-AS cells. CONCLUSIONS: In this study, we demonstrated the programmed expression of full-length BMCC1 in human NB cells undergoing DNA damage-induced apoptosis. The elucidation of the molecular mechanisms controlling the regulation of BMCC1 during apoptosis initiated by DNA damage provides useful information for understanding drug resistance of tumor cells and spontaneous regression of NB.

Screening of a library of traditional Chinese medicines to identify anti-malarial compounds and extracts.

BACKGROUND: Malaria is a major infectious disease in the world. In 2015, approximately 212 million people were infected and 429,000 people were killed by this disease. Plasmodium falciparum, which causes falciparum malaria, is becoming resistant to artemisinin (ART) in Southeast Asia; therefore, new anti-malarial drugs are urgently needed. Some excellent anti-malarial drugs, such as quinine or ART, were originally obtained from natural plants. Hence, the authors screened a natural product library comprising traditional Chinese medicines (TCMs) to identify compounds/extracts with anti-malarial effects. METHODS: The authors performed three assays: a malaria growth inhibition assay (GIA), a cytotoxicity assay, and a malaria stage-specific GIA. The malaria GIA revealed the anti-malarial ability and half-maximal inhibitory concentrations (IC50) of the natural products, whereas the malaria stage-specific GIA revealed the point in the malaria life cycle where the products exerted their anti-malarial effects. The toxicity of the products to the host cells was evaluated with the cytotoxicity assay. RESULTS: Four natural compounds (berberine chloride, coptisine chloride, palmatine chloride, and dehydrocorydaline nitrate) showed strong anti-malarial effects (IC50 < 50 nM), and low cytotoxicity (cell viability > 90%) using P. falciparum 3D7 strain. Two natural extracts (Phellodendri cortex and Coptidis rhizoma) also showed strong antiplasmodial effects (IC50 < 1 µg/ml), and low cytotoxicity (cell viability > 80%). These natural products also demonstrated anti-malarial capability during the trophozoite and schizont stages of the malaria life cycle. CONCLUSIONS: The authors identified four compounds (berberine chloride, coptisine chloride, palmatine chloride, and dehydrocorydaline nitrate) and two extracts (Phellodendri cortex and Coptidis rhizoma) with anti-malarial activity, neither of which had previously been described. The IC50 values of the compounds were comparable to that of chloroquine and better than that of pyrimethamine. These compounds and extracts derived from TCMs thus show promise as potential future anti-malarial drugs.

Rhodium-Catalyzed Synthesis and Optical Properties of Silicon-Bridged Arylpyridines.

A convergent and regioselective synthesis of silicon-bridged 4-arylpyridines has been developed through a rhodium-catalyzed [2+2+2] cycloaddition of silicon-containing diynes with nitriles. The absorption and emission properties of these compounds have been examined and could be tuned by varying the substituent on the benzene ring, as well as through the protonation or alkylation of the nitrogen atom on the pyridine ring. A catalytic asymmetric synthesis of silicon-centered axially chiral spirocyclic derivatives has also been achieved with high enantioselectivity by using a newly modified MeO-MOP (MeO-MOP=2-(diphenylphosphino)-2'-methoxy-1,1'-binaphthyl) derivative as the chiral ligand. These spirocyclic compounds were found to be CPL-active (CPL=circularly polarized luminescence), representing the first CPL-active compounds based on the chirality at silicon.

Transcriptional regulation of BMCC1 mediated by E2F1 in neuroblastoma cells.

BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1)/PRUNE2 is highly expressed in patients with favorable neuroblastoma (NB), encoding a multifunctional scaffold protein that modulates several signaling networks including RhoA and AKT pathways. Accumulating evidence suggests that BMCC1 acts as a tumor-suppressor. In this study, we addressed molecular mechanism underlying transcriptional regulation of BMCC1 in NBs. We found that transcription factor E2F1 was recruited to E2F-binding site in the promoter region of BMCC1 gene. Indeed, overexpression of E2F1 resulted in an increase in the expression level of BMCC1 in NB cell lines. On the other hand, knockdown of E2F1 in NB cells yielded down-regulation of BMCC1. Also, we showed that BMCC1 and E2F1 were simultaneously induced at G1 to S phase transition. Therefore, we conclude that E2F1 directly facilitated BMCC1 transcription. Taking together, these results suggest that BMCC1 induced by E2F1 acts as a tumor suppressor through its pro-apoptotic function, resulted in favorable prognosis of NB.

Disease severity is associated with differential gene expression at the early and late phases of infection in nonhuman primates infected with different H5N1 highly pathogenic avian influenza viruses.

UNLABELLED: Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome. IMPORTANCE: Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

F1Fo-ATPase, F-type proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding.

The identification of host factors involved in virus replication is important to understand virus life cycles better. Accordingly, we sought host factors that interact with the influenza viral nonstructural protein 2 by using coimmunoprecipitation followed by mass spectrometry. Among proteins associating with nonstructural protein 2, we focused on the ß subunit of the F1Fo-ATPase, which received a high probability score in our mass spectrometry analysis. The siRNA-mediated down-regulation of the ß subunit of the F1Fo-ATPase reduced influenza virion formation and virus growth in cell culture. We further found that efficient influenza virion formation requires the ATPase activity of F1Fo-ATPase and that plasma membrane-associated, but not mitochondrial, F1Fo-ATPase is important for influenza virion formation and budding. Hence, our data identify plasma membrane-associated F1Fo-ATPase as a critical host factor for efficient influenza virus replication.

Virulence determinants of pandemic A(H1N1)2009 influenza virus in a mouse model.

A novel swine-origin H1N1 influenza virus [A(H1N1)pdm09 virus] caused the 2009 influenza pandemic. Most patients exhibited mild symptoms similar to seasonal influenza, but some experienced severe clinical signs and, in the worst cases, died. Such differences in symptoms are generally associated with preexisting medical conditions, but recent reports indicate the possible involvement of viral factors in clinical severity. To better understand the mechanism of pathogenicity of the A(H1N1)pdm09 virus, here, we compared five viruses that are genetically similar but were isolated from patients with either severe or mild symptoms. In a mouse model, A/Norway/3487/2009 (Norway3487) virus exhibited greater pathogenicity than did A/Osaka/164/2009 (Osaka164) virus. By exploiting reassortant viruses between these two viruses, we found that viruses possessing the hemagglutinin (HA) gene of Norway3487 in the genetic background of Osaka164 were more pathogenic in mice than other reassortant viruses, indicating a role for HA in the high virulence of Norway3487 virus. Intriguingly, a virus possessing HA, NA, and NS derived from Norway3487 exhibited greater pathogenicity in mice in concert with PB2 and PB1 derived from Osaka164 than did the parental Norway3487 virus. These findings demonstrate that reassortment between A(H1N1)pdm09 viruses can lead to increased pathogenicity and highlight the need for continued surveillance of A(H1N1)pdm09 viruses.

Lambda-carrageenan treatment exacerbates the severity of cerebral malaria caused by Plasmodium berghei ANKA in BALB/c mice.

BACKGROUND: There is an urgent need to develop and test novel compounds against malaria infection. Carrageenans, sulphated polysaccharides derived from seaweeds, have been previously shown to inhibit Plasmodium falciparum in vitro. However, they are inflammatory and alter the permeability of the blood-brain barrier, raising concerns that their use as a treatment for malaria could lead to cerebral malaria (CM), a severe complication of the disease. In this work, the authors look into the effects of the administration of λ-carrageenan to the development and severity of CM in BALB/c mice, a relatively non-susceptible model, during infection with the ANKA strain of Plasmodium berghei. METHODS: Five-week-old female BALB/c mice were infected with P. berghei intraperitoneally. One group was treated with λ-carrageenan (PbCGN) following the 4-day suppressive test protocol, whereas the other group was not treated (PbN). Another group of healthy BALB/c mice was similarly given λ-carrageenan (CGN) for comparison. The following parameters were assessed: parasitaemia, clinical signs of CM, and mortality. Brain and other vital organs were collected and examined for gross and histopathological lesions. Evans blue dye assays were employed to assess blood-brain barrier integrity. RESULTS: Plasmodium berghei ANKA-infected BALB/c mice treated with λ-carrageenan died earlier than those that received no treatment. Histopathological examination revealed that intracerebral haemorrhages related to CM were present in both groups of infected BALB/c mice, but were more numerous in those treated with λ-carrageenan than in mock-treated animals. Inflammatory lesions were also observed only in the λ-carrageenan-treated mice. These observations are consistent with the clinical signs associated with CM, such as head tilt, convulsions, and coma, which were observed only in this group, and may account for the earlier death of the mice. CONCLUSION: The results of this study indicate that the administration of λ-carrageenan exacerbates the severe brain lesions and clinical signs associated with CM in BALB/c mice infected with P. berghei ANKA.

Molecular mechanisms underlying oseltamivir resistance mediated by an I117V substitution in the neuraminidase of subtype H5N1 avian influenza A viruses.

BACKGROUND: The neuraminidase (NA) inhibitor oseltamivir is widely used to treat patients infected with influenza viruses. An Ile-to-Val change at position 117 in influenza A virus subtype H5N1 NA (NA-I117V) confers a reduction in susceptibility to oseltamivir carboxylate. However, the in vivo relevance and molecular basis of the decreased sensitivity mediated by this mutation are poorly understood. METHODS: We created single-point-mutant viruses with 3 genetically different backgrounds (ie, 1 belonging to clade 1 and 2 belonging to clade 2.3.4) and evaluated the effects of the I117V mutation on oseltamivir susceptibility in vitro, in vivo, and in silico. RESULTS: The NA-I117V mutation conferred a slight reduction in susceptibility to oseltamivir in vitro (1.3- to 6.3-fold changes), although it did not substantially compromise NA enzymatic activity. Mice infected with I117V virus exhibited reduced susceptibility to oseltamivir and decreased survival in 2 of 3 virus pairs tested. Molecular dynamics simulations revealed that I117V caused the loss of hydrogen bonds between an arginine at position 118 and the carboxyl group of oseltamivir, leading to a lower binding affinity for oseltamivir. CONCLUSIONS: Our findings provide new insight into the mechanism of NA-I117V-mediated oseltamivir resistance in highly pathogenic H5N1 avian influenza viruses.

Differences in cytokine production in human macrophages and in virulence in mice are attributable to the acidic polymerase protein of highly pathogenic influenza A virus subtype H5N1.

BACKGROUND: The pathogenesis of influenza A virus subtype H5N1 (hereafter, "H5N1") infection in humans is not completely understood, although hypercytokinemia is thought to play a role. We previously reported that most H5N1 viruses induce high cytokine responses in human macrophages, whereas some H5N1 viruses induce only a low level of cytokine production similar to that induced by seasonal viruses. METHODS: To identify the viral molecular determinants for cytokine induction of H5N1 viruses in human macrophages, we generated a series of reassortant viruses between the high cytokine inducer A/Vietnam/UT3028II/03 clone 2 (VN3028IIcl2) and the low inducer A/Indonesia/UT3006/05 (IDN3006) and evaluated cytokine expression in human macrophages. RESULTS: Viruses possessing the acidic polymerase (PA) gene of VN3028IIcl2 exhibited high levels of hypercytokinemia-related cytokine expression in human macrophages, compared with IDN3006, but showed no substantial differences in viral growth in these cells. Further, the PA gene of VN3028IIcl2 conferred enhanced virulence in mice. CONCLUSIONS: These results demonstrate that the PA gene of VN3028IIcl2 affects cytokine production in human macrophages and virulence in mice. These findings provide new insights into the cytokine-mediated pathogenesis of H5N1 infection in humans.

Self-administered generational surveys combine with genetic analysis to reveal foundations of depression in Japanese adults.

BACKGROUND: Major depressive disorder is a prevalent psychiatric illness characterized by mood disturbances and influenced by various environmental and genetic factors, yet its etiology remains largely unknown. METHODS: We profiled a self-reported depressive population in Japan with a focus on sociodemographic background, lifestyle, comorbidities, and genetic background, using data from two cohorts, a population-based cohort and a three-generation cohort, recruited by the Tohoku Medical Megabank Organization until December 2021. RESULTS: Our findings revealed that depression in the Japanese population is strongly associated with certain sociocultural features prevalent in Japan, such as social isolation, neuroticism, and introversion, as well as with well-known risk factors that include age and gender. Environmental factors related to the Great East Japan Earthquake, considered as cohort characteristics, were also strongly associated with the onset of depression. Moreover, using GWAS analysis of whole-genome sequencing data, we identified novel candidate genetic risk variants located on chromosomes 21 and 22 that are associated with depression in Japanese individuals; further validation of these risk variants is warranted. LIMITATIONS: Our study has limitations, including uncertain clinical relevance resulting from the use of self-reported questionnaires for depression assessment. Additionally, the cohort exhibited a population bias, with greater representation of women than men. CONCLUSIONS: Our results provide holistic insights into depression risk factors in Japanese adults, although their associations with depression are correlations. This supports the idea that targeted interventions and individualized approaches are important for addressing depression in the Japanese population.

Recurrent pulmonary hypertension after balloon pulmonary angioplasty for inoperable chronic thromboembolic pulmonary hypertension.

BACKGROUND: Balloon pulmonary angioplasty improves the hemodynamics of patients with inoperable chronic thromboembolic pulmonary hypertension; however, the clinical impact of recurrent pulmonary hypertension after balloon pulmonary angioplasty remains unclear. METHODS: We retrospectively reviewed 262 consecutive patients with chronic thromboembolic pulmonary hypertension who underwent balloon pulmonary angioplasty between July 2009 and December 2020; 158 (65 ± 12 years; males, 20%; median follow-up period, 45 [26, 66] months) with follow-up right heart catheterization and no residual pulmonary hypertension were included. Recurrent pulmonary hypertension was defined as mean pulmonary arterial pressure <25 mm Hg at the first evaluation after balloon pulmonary angioplasty and ≥25 mm Hg at follow-up evaluation requiring additional treatment with balloon pulmonary angioplasty or pulmonary vasodilators. RESULTS: Recurrent pulmonary hypertension was observed in 11 patients; the state occupation probability of recurrence at 5 years was 9.0% (95% confidence interval: 5.0%-18.9%). Only 1 case (0.6%) of recurrent pulmonary hypertension showed vascular restenosis and reocclusion of previously treated lesions, with more significant hemodynamic and exercise capacity deterioration than the other cases. Additional treatments for recurrent pulmonary hypertension (balloon pulmonary angioplasty in 9 patients, pulmonary vasodilators in 4 patients) improved the mean pulmonary arterial pressure from 27 [26, 29] to 22 [19, 23] mm Hg (p < 0.01). Recurrence had a low probability of transitioning to death in an illness-death model. No specific risk factors for recurrent pulmonary hypertension were identified. CONCLUSIONS: Symptomatic recurrent pulmonary hypertension due to vascular restenosis or reocclusion after balloon pulmonary angioplasty was extremely rare. Most cases of recurrent pulmonary hypertension were mild, did not worsen clinically, and had favorable prognoses.

Apalutamide and Goserelin for Androgen Receptor-Positive Salivary Gland Carcinoma: A Phase 2 Nonrandomized Clinical Trial, YATAGARASU.

PURPOSE: To assess efficacy and safety of apalutamide plus goserelin for androgen receptor (AR)-positive, unresectable or recurrent/metastatic salivary gland carcinoma (URM-SGC). PATIENTS AND METHODS: This was an open-label, single-arm, multicenter phase II study for patients with AR-positive URM-SGC. The primary endpoint was the overall response rate (ORR) by an independent central radiology review (ICRR) in the first 24 response evaluable patients who had been observed at least 24 weeks from study initiation (primary RE patients). The efficacy was to be declared when at least 8 of the 24 primary RE patients responded. RESULTS: 31 patients were enrolled. In the first 24 primary RE patients with a median follow-up of 7.4 months, confirmed ORR by ICRR was 25.0% (6/24 patients; 95%CI: 9.8%-46.7%; P =0.11 (one-sided)), which did not meet the predefined criteria of efficacy. Clinical benefit rate (ORR + rate of stable disease for at least 24 weeks) and median progression-free survival were 50.0% and 7.4 months, respectively. Both median duration of response and overall survival were not reached. Exploratory analyses showed a better ORR of 54.5% (6/11) in patients with AR-positivity ≥ 70% and no history of prior systemic therapy. Grade 3 or higher treatment-emergent adverse events were reported in 35.5% (11/31), which included skin rash, anemia, leukopenia, and cancer pain. CONCLUSIONS: Although this study did not meet the predefined efficacy criteria, apalutamide plus goserelin showed clinically meaningful efficacy in a subset of patients with AR-positive SGC and safety consistent with prior experience in prostate cancer.

Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion.

Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.

Avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques.

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.

Characterization of oseltamivir-resistant 2009 H1N1 pandemic influenza A viruses.

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.