Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte‐derived dendritic cells

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro‐inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte‐derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF‐α, ‐β and ‐γ were significantly upregulated in the H5N1 group. Pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐18) were highly upregulated in early mid (IL‐1), and late (IL‐6) phases of H5N1 virus infection. IL‐8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus‐induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV‐infected cells.     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

Influenza A: From highly pathogenic H5N1 to pandemic 2009 H1N1. Epidemiology and clinical features

The last decade has seen the emergence of two new influenza A subtypes and they have become a cause of concern for the global community. These are the highly pathogenic H5N1 influenza A virus (H5N1) and the Pandemic 2009 influenza H1N1 virus. Since 2003 the H5N1 virus has caused widespread disease and death in poultry, mainly in south East Asia and Africa. In humans the number of cases infected with this virus is few but the mortality has been about 60%. Most patients have presented with severe pneumonia and acute respiratory distress syndrome. The second influenza virus, the pandemic H1N1 2009, emerged in Mexico in March this year. This virus acquired the ability for sustained human to human spread and within a few months spread throughout the world and infected over 4 lakh individuals. The symptoms of infection with this virus are similar to seasonal influenza but it currently affecting younger individuals more often. Fortunately the mortality has been low. Both these new influenza viruses are currently circulating and have different clinical and epidemiological characteristics.     

人感染H7亚型禽流感事件

本研究综述了自1959年以来国内外发生的人感染H7亚型禽流感事件。大多数是在家禽爆发禽流感期间,农场工人在处置感染鸡群过程中被暴露而感染;也有曾接触活禽或曾到过活禽市场而感染;有经禽流感病毒致病的哺乳动物（海豹）感染于人或实验室感染（事故）所致。引起人感染的H7亚型中已知有H7N2、H7N3、H7N7以及2013年在中国发现的新的致病亚型H7N9。H7N2、H7N3、H7N7感染以结膜炎为主,大多为轻症;而H7N9感染以严重的呼吸道感染为特征,表现为重症肺炎,呼吸窘迫综合症,病死率高达33.6%。     

Viral replication and innate host responses in primary human alveolar epithelial cells and alveolar macrophages infected with influenza H5N1 and H1N1 viruses

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocyte-derived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.     

Genome Wide Host Gene Expression Analysis in Chicken Lungs Infected with Avian Influenza Viruses

The molecular pathogenesis of avian influenza infection varies greatly with individual bird species and virus strain. The molecular pathogenesis of the highly pathogenic avian influenza virus (HPAIV) or the low pathogenic avian influenza virus (LPAIV) infection in avian species remains poorly understood. Thus, global immune response of chickens infected with HPAI H5N1 (A/duck/India/02CA10/2011) and LPAI H9N2 (A/duck/India/249800/2010) viruses was studied using microarray to identify crucial host genetic components responsive to these infection. HPAI H5N1 virus induced excessive expression of type I IFNs (IFNA and IFNG), cytokines (IL1B, IL18, IL22, IL13, and IL12B), chemokines (CCL4, CCL19, CCL10, and CX3CL1) and IFN stimulated genes (OASL, MX1, RSAD2, IFITM5, IFIT5, GBP 1, and EIF2AK) in lung tissues. This dysregulation of host innate immune genes may be the critical determinant of the severity and the outcome of the influenza infection in chickens. In contrast, the expression levels of most of these genes was not induced in the lungs of LPAI H9N2 virus infected chickens. This study indicated the relationship between host immune genes and their roles in pathogenesis of HPAIV infection in chickens.     

Early induction of interleukin-5 and peripheral eosinophilia in acute pneumonia in Japanese children infected by pandemic 2009 influenza A in the Tokyo area

A novel influenza A (2009 H1N1) virus has led to a worldwide pandemic. A significant number of patients with pneumonia have been reported, although its pathogenesis remains to be elucidated. To determine its pathogenesis, we evaluated serum interleukin (IL)-5 and peripheral eosinophil counts in patients with acute pneumonia caused by the 2009 H1N1 virus. During the period from October to December 2009, 40 patients with laboratory-confirmed 2009 H1N1 pneumonia were under investigation. Their mean age at presentation was 6.8 years. The most characteristic finding was the early development of hypoxemic respiratory distress in the first 24 hr after the onset of fever. Bronchial mucous plugs included eosinophils in addition to neutrophils, even in patients without allergies. Serum IL-5 levels were elevated in 20 out of 24 patients (83%) whose samples were obtained in the first 24 hr after the onset of fever (26.5 ± 20.1 pg/mL), independent of the presence of underlying allergies. In contrast, induction of IL-5 was not documented in sera from eight patients with laboratory-confirmed 2009 H1N1 virus who developed neurological complications, but without lower respiratory infection (2.1 ± 0.7 pg/mL, P < 0.001 vs acute pneumonia). Peripheral eosinophilia was characteristic in acute pneumonia, but not in patients without a lower respiratory infection. There was a marked difference in the induction of IL-5 in 2009 H1N1 patients who developed acute pneumonia, compared with those without a lower respiratory infection. IL-5 may play a role in the early phase of acute pneumonia caused by the 2009 H1N1 virus in Japanese children.     

Comparison of Temporal and Spatial Dynamics of Seasonal H3N2, Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Virus Infections in Ferrets

Humans may be infected by different influenza A viruses-seasonal, pandemic, and zoonotic-which differ in presentation from mild upper respiratory tract disease to severe and sometimes fatal pneumonia with extra-respiratory spread. Differences in spatial and temporal dynamics of these infections are poorly understood. Therefore, we inoculated ferrets with seasonal H3N2, pandemic H1N1 (pH1N1), and highly pathogenic avian H5N1 influenza virus and performed detailed virological and pathological analyses at time points from 0.5 to 14 days post inoculation (dpi), as well as describing clinical signs and hematological parameters. H3N2 infection was restricted to the nose and peaked at 1 dpi. pH1N1 infection also peaked at 1 dpi, but occurred at similar levels throughout the respiratory tract. H5N1 infection occurred predominantly in the alveoli, where it peaked for a longer period, from 1 to 3 dpi. The associated lesions followed the same spatial distribution as virus infection, but their severity peaked between 1 and 6 days later. Neutrophil and monocyte counts in peripheral blood correlated with inflammatory cell influx in the alveoli. Of the different parameters used to measure lower respiratory tract disease, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups. There was extra-respiratory spread to more tissues-including the central nervous system-for H5N1 infection than for pH1N1 infection, and to none for H3N2 infection. This study shows that seasonal, pandemic, and zoonotic influenza viruses differ strongly in the spatial and temporal dynamics of infection in the respiratory tract and extra-respiratory tissues of ferrets.     

H7N9禽流感病毒与其他流感病毒对雪貂致病性与传播力的比较

目的针对2013年3月中国爆发的人感染H7N9禽流感病毒,在雪貂体内进行致病性及传播力的研究,并与甲型H1N1流感病毒、H5N1禽流感病毒进行比较。方法对新发H7N9毒株、甲型H1N1流感病毒、H5N1禽流感病毒感染雪貂后的临床症状、体征,呼吸道排毒情况,组织病理学变化等进行评价和比较,并对H7N9毒株在雪貂群体中的传播力进行研究。结果雪貂模型的临床症状、死亡率、病毒传播以及组织病理学分析显示:H7N9病毒的致病性低于H5N1,与2009年起源于北美的甲型H1N1流感病毒相当。新发H7N9禽流感病毒可以在雪貂的呼吸道、心脏、肝脏以及嗅球进行复制。值得注意的是H7N9禽流感可以通过飞沫在雪貂间进行低水平的传播,并且在传播过程中,病毒基因组内有多个位点的氨基酸发生了替换。结论 H7N9禽流感病毒对雪貂的致病性较H5N1禽流感病毒低,与甲型H1N1流感病毒对雪貂的致病性相当,H7N9禽流感病毒可在雪貂间进行传播。     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment.     

Mammary transmission of caprine arthritis encephalitis virus: a 3D model for in vitro study

Transmission of Caprine Arthritis Encephalitis virus (CAEV) from the mother to offspring is principally mediated by infected cells from colostrum and milk. The infection of the dam is often sub-clinical, and results in increased cellularity of milk, sometimes exacerbated by bacterial co-infections. Although monocytes are the major viral host cells, several other cell types, including epithelial mammary cells, fibroblasts and endothelial cells show low levels of in vivo infection. In vitro, however, all phenotypes of mammary gland cells are individually highly sensitive to CAEV infection. This suggests that local mechanisms act to control viral expression. Our goal is to analyse the mechanisms regulating local virus infection, including the physiological status of the mammary gland and bacterial co-infections. In this work, we present the development of a model for the in vitro reconstitution of mammary gland tissue using 3D cultures in Matrigel. Mononuclear cells from the blood are added to the 3D cultures in vitro. In these experimental conditions, the mammary cells spontaneously organize into mammospheres. Blood leucocytes migrate into the culture gel, and localize particularly at the periphery of the mammospheres. Mammospheres were susceptible to infection in vitro by CAEV, as shown by a cytopathic effect and expression of late CAEV antigen p30. This model will allow the in vitro study of virus expression, transfer of infection to mammary gland cells and interactions between the mammary gland cells, infected monocytes and immunocompetent cells. It will allow the study of mechanisms participating in the control of passage of pathogens into milk, according to the physiological and CAEV-infection status of the animal, microenvironment and the presence of bacterial co-infections.     

Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets

How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.     

Duvira Antarctic polysaccharide inhibited H1N1 influenza virus-induced apoptosis through ROS mediated ERK and STAT-3 signaling pathway

Background

The H1N1 influenza virus causes acute respiratory tract infection, and its clinical symptoms are very similar to those of ordinary influenza. The disease develops rapidly. If the flu is not treated, complications such as pneumonia, respiratory failure, and multiple organ damage can occur, resulting in a high fatality rate. Influenza virus mutates rapidly. At present, there is no specific drug for H1N1, so it is an urgent need for clinical care to find new drugs to treat H1N1.

Materials and methods

The polysaccharide derived from Durvillaea Antarctica green algae has a certain antiviral effect. In this study, the results of CCK-8, apoptosis cycle detection, JC-1 and Western blotting proved that Duvira Antarctic polysaccharide (DAPP) has the ability to inhibit H1N1 infection.

Results

CCK-8 test showed that the DAPP with concentration at 32 μg/mL had no toxicity to MDCK cells. In addition, DAPP reduced cell apoptosis by inhibiting the ERK signaling pathway. Meanwhile, DAPP could increase the expression of STAT3 and significantly inhibited proinflammatory cytokines.

Conclusions

In summary, these results suggested that DAPP may be potential with the ability to resist the H1N1 influenza virus.

     

Cytokine and chemokine responses in pediatric patients with severe pneumonia associated with pandemic A/H1N1/2009 influenza virus

Severe pneumonia and leukocytosis are characteristic, frequently observed, clinical findings in pediatric patients with pandemic A/H1N1/2009 influenza virus infection. The aim of this study was to elucidate the role of cytokines and chemokines in complicating pneumonia and leukocytosis in patients with pandemic A/H1N1/2009 influenza virus infection. Forty‐seven patients with pandemic A/H1N1/2009 influenza virus infection were enrolled in this study. Expression of interleukin (IL)‐10 (P = 0.027) and IL‐5 (P = 0.014) was significantly greater in patients with pneumonia than in those without pneumonia. Additionally, serum concentrations of interferon‐γ (P = 0.009), tumor necrosis factor‐α (P = 0.01), IL‐4 (P = 0.024), and IL‐2 (P = 0.012) were significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without neutrophilic leukocytosis. Of the five serum chemokine concentrations assessed, only IL‐8 was significantly lower in pneumonia patients with neutrophilic leukocytosis than in those without leukocytosis (P = 0.001). These cytokines and chemokines may play important roles in the pathogenesis of childhood pneumonia associated with A/H1N1/2009 influenza virus infection.     

Novel H7N9 Influenza Virus Shows Low Infectious Dose,High Growth Rate,and Efficient Contact Transmission in the Guinea Pig Model

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.     

Ocular tropism of influenza A viruses: identification of H7 subtype-specific host responses in human respiratory and ocular cells

Highly pathogenic avian influenza (HPAI) H7 virus infection in humans frequently results in conjunctivitis as a major symptom. However, our understanding of what properties govern virus subtype-specific tropism, and of the host responses responsible for eliciting ocular inflammation and pathogenicity following influenza virus infection, are not well understood. To study virus-host interactions in ocular tissue, we infected primary human corneal and conjunctival epithelial cells with H7, H5, and H1 subtype viruses. We found that numerous virus subtypes were capable of infecting and replicating in multiple human ocular cell types, with the highest titers observed with highly pathogenic H7N7 and H5N1 viruses. Similar patterns of proinflammatory cytokine and chemokine production following influenza virus infection were observed in ocular and respiratory cells. However, primary ocular cells infected with HPAI H7N7 viruses were found to have elevated levels of interleukin-1β (IL-1β), a cytokine previously implicated in ocular disease pathology. Furthermore, H7N7 virus infection of corneal epithelial cells resulted in enhanced and significant increases in the expression of genes related to NF-κB signal transduction compared with that after H5N1 or H1N1 virus infection. The differential induction of cytokines and signaling pathways in human ocular cells following H7 virus infection marks the first association of H7 subtype-specific host responses with ocular tropism and pathogenicity. In particular, heightened expression of genes related to NF-κB-mediated signaling transduction following HPAI H7N7 virus infection in primary corneal epithelial cells, but not respiratory cells, identifies activation of a signaling pathway that correlates with the ocular tropism of influenza viruses within this subtype.     

Pathogenesis and transmission of triple-reassortant swine H1N1 influenza viruses isolated before the 2009 H1N1 pandemic

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.     

The beginning and ending of a respiratory viral pandemic-lessons from the Spanish flu

The COVID-19 pandemic goes into its third year and the world population is longing for an end to the pandemic. Computer simulations of the future development of the pandemic have wide error margins and predictions on the evolution of new viral variants of SARS-CoV-2 are uncertain. It is thus tempting to look into the development of historical viral respiratory pandemics for insight into the dynamic of pandemics. The Spanish flu pandemic of 1918 caused by the influenza virus H1N1 can here serve as a potential model case. Epidemiological observations on the shift of influenza mortality from very young and old subjects to high mortality in young adults delimitate the pandemic phase of the Spanish flu from 1918 to 1920. The identification and sequencing of the Spanish flu agent allowed following the H1N1 influenza virus after the acute pandemic phase. During the 1920s H1N1 influenza virus epidemics with substantial mortality were still observed. As late as 1951, H1N1 strains of high virulence evolved but remained geographically limited. Until 1957, the H1N1 virus evolved by accumulation of mutations (‘antigenic drift’) and some intratypic reassortment. H1N1 viruses were then replaced by the pandemic H2N2 influenza virus from 1957, which was in 1968 replaced by the pandemic H3N2 influenza virus; both viruses were descendants from the Spanish flu agent but showed the exchange of entire gene segments (‘antigenic shift’). In 1977, H1N1 reappeared from an unknown source but caused only mild disease. However, H1N1 achieved again circulation in the human population and is now together with the H3N2 influenza virus an agent of seasonal influenza winter epidemics.