The role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice

Background

Neutrophils have been shown to play a role in host defence against highly virulent and mouse-adapted strains of influenza virus, however it is not clear if an effective neutrophil response is an important factor moderating disease severity during infection with other virus strains. In this study, we have examined the role of neutrophils during infection of mice with influenza virus strain HKx31, a virus strain of the H3N2 subtype and of moderate virulence for mice, to determine the role of neutrophils in the early phase of infection and in clearance of influenza virus from the respiratory tract during the later phase of infection.

Methods

The anti-Gr-1 monoclonal antibody (mAb) RB6-8C5 was used to (i) identify neutrophils in the upper (nasal tissues) and lower (lung) respiratory tract of uninfected and influenza virus-infected mice, and (ii) deplete neutrophils prior to and during influenza virus infection of mice.

Results

Neutrophils were rapidly recruited to the upper and lower airways following influenza virus infection. We demonstrated that use of mAb RB6-8C5 to deplete C57BL/6 (B6) mice of neutrophils is complicated by the ability of this mAb to bind directly to virus-specific CD8⁺ T cells. Thus, we investigated the role of neutrophils in both the early and later phases of infection using CD8⁺ T cell-deficient B6.TAP^-/- mice. Infection of B6.TAP^-/- mice with a low dose of influenza virus did not induce clinical disease in control animals, however RB6-8C5 treatment led to profound weight loss, severe clinical disease and enhanced virus replication throughout the respiratory tract.

Conclusion

Neutrophils play a critical role in limiting influenza virus replication during the early and later phases of infection. Furthermore, a virus strain of moderate virulence can induce severe clinical disease in the absence of an effective neutrophil response.     

Inhibition of lectin-mediated innate host defences in vivo modulates disease severity during influenza virus infection

Host-mediated recognition of mannose-rich glycans on the surface of pathogens represents an ancient mechanism of innate immune defence. In this study, we demonstrate that the virus strains that differ in the degree of N-linked glycosylation on the globular head of their hemagglutinin glycoprotein also differed in their (i) sensitivity to neutralization by a mannose-specific lectin in mouse lung fluids and (ii) ability to infect (and, therefore, to be destroyed) by airway macrophages. Virus strain BJx109 (H3N2), but not PR8 (H1N1), was sensitive to neutralization by mouse lung fluids and infected airway macrophages efficiently in vitro and these antiviral activities were blocked by mannan, a complex polymer of mannose residues. Although intranasal (i.n.) infection of mice with PR8 led to severe disease and mortality, mice infected with an equivalent dose of BJx109 displayed no signs of disease. However, i.n. treatment of BJx109-infected mice with mannan led to viral pneumonia, severe disease and death characterized by excessive virus replication, pulmonary inflammation, vascular leak and lung edema. Thus, when mannose-specific innate defences were inhibited in vivo, virus strain BJx109 induced severe viral pneumonia similar to that of PR8. Together, these findings highlight the importance of N-linked glycans as a target for recognition and destruction of influenza viruses by the innate immune system. Moreover, soluble and cell-associated lectins coordinate to modulate disease severity following influenza virus infection of mice.     

Critical Role of Airway Macrophages in Modulating Disease Severity during Influenza Virus Infection of Mice

Airway macrophages provide a first line of host defense against a range of airborne pathogens, including influenza virus. In this study, we show that influenza viruses differ markedly in their abilities to infect murine macrophages in vitro and that infection of macrophages is nonproductive and no infectious virus is released. Virus strain BJx109 (H3N2) infected macrophages with high efficiency and was associated with mild disease following intranasal infection of mice. In contrast, virus strain PR8 (H1N1) was poor in its ability to infect macrophages and highly virulent for mice. Depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in BJx109-infected mice but did not modulate disease severity in PR8-infected mice. The severe disease observed in macrophage-depleted mice infected with BJx109 was associated with exacerbated virus replication in the airways, leading to severe airway inflammation, pulmonary edema, and vascular leakage, indicative of lung injury. Thymic atrophy, lymphopenia, and dysregulated cytokine and chemokine production were additional systemic manifestations associated with severe disease. Thus, airway macrophages play a critical role in limiting lung injury and associated disease caused by BJx109. Furthermore, the inability of PR8 to infect airway macrophages may be a critical factor contributing to its virulence for mice.Airway macrophages (Mφ) (AM), located at the interphase between air and lung tissue, provide the first line of defense following inhalation of airborne pathogens, including influenza viruses. In addition to phagocytosis of virions and virus-infected cells (16, 24), infection of AM represents an early event in recognition of the virus by the innate immune system. Following intranasal (i.n.) infection of mice, influenza virus replicates productively in type II epithelial cells lining the respiratory tract. Murine Mφ are also susceptible to influenza virus infection and viral proteins are produced, but replication is abortive and infectious progeny are not released (52, 69), although recent studies suggest limited release from mouse Mφ exposed to highly pathogenic H1N1 and H5N1 viruses (44). Following exposure to influenza virus, Mφ synthesize and release proinflammatory cytokines and alpha/beta interferon (26, 27, 45, 55), which further limit viral replication and spread within the respiratory tract. Inflammatory responses in the airways must be tightly regulated to ensure rapid virus clearance while avoiding excessive or chronic inflammation that may damage the delicate tissue-air interface.Liposome-encapsulated dichloromethylene diphosphonate (clodronate or CL2-MDP) is taken up by phagocytic Mφ and accumulates in the cytosol, resulting in Mφ death and depletion (66). Administration of clodronate liposomes (CL-LIP) has been widely used to selectively deplete airway Mφ in mouse models without affecting circulating monocytes (for examples, see references 4, 6, 8, 36, 47, 64, and 71). While CL-LIP has been used predominantly in rodent models of infection, it is noteworthy that CL-LIP treatment of pigs, a natural host of influenza virus, resulted in enhanced morbidity and mortality following infection with a human H1N1 subtype virus (30). In murine studies, depletion of airway Mφ prior to influenza virus infection led to increased cytotoxic CD8⁺ T-cell responses in the lungs of virus-infected mice (71); however, treatment with CL-LIP 48 h after infection was associated with impaired CD8⁺ T-cell responses (41). Furthermore, CL-LIP treatment prior to intranasal infection of mice with a recombinant virus bearing the surface glycoproteins of the 1918 pandemic strain led to exacerbated disease and mortality (64). Treatment of mice with CL-LIP has been associated with enhanced virus replication (41, 64, 71); however, the mechanisms by which airway Mφ initiate and modulate inflammatory responses and disease after influenza virus infection have not been fully elucidated.We observed in a previous study that influenza A virus strains show marked differences in their abilities to infect murine Mφ in vitro and implicated the Mφ mannose receptor (MMR) (CD206), a C-type lectin (46), in infectious virus entry (48). Virus strain BJx109, a reassortant virus bearing the surface glycoproteins of A/Beijing/353/89 (H3N2) and internal components derived from A/PR/8/34 (H1N1) (PR8) bears a highly glycosylated hemagglutinin (HA) molecule and was shown to infect Mφ to high levels, while the HA of PR8 is poorly glycosylated and the virus infected Mφ very poorly. In the current study, we demonstrate that intranasal infection of mice with BJx109 leads to mild clinical disease, while infection with an equivalent dose of PR8 leads to severe disease and rapid death. Depletion of airway Mφ by intranasal administration of CL-LIP prior to and during infection with influenza viruses had little effect on the course of PR8 infection; however, BJx109 infection of Mφ-depleted animals led to severe disease and death. Severe disease was associated with enhanced virus replication, severe airway inflammation, and pulmonary edema and vascular leakage, indicative of lung injury. Together, these data demonstrate that airway Mφ play a critical role in moderating disease severity during BJx109 infection. Furthermore, they suggest that the ability of PR8 to evade infectious uptake by airway Mφ is likely to be an important factor contributing to its virulence for mice.     

Involvement of the mannose receptor in infection of macrophages by influenza virus

Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774. The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family. Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus. In competitive binding studies, the binding of (125)I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains. The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages. Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan. A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of D-mannose, which down-modulated mannose receptor expression. Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.     

Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.     

Gene expression changes in the host response between resistant and susceptible inbred mouse strains after influenza A infection

Inbred mouse strains exhibit differences in susceptibility to influenza A infections. However, the molecular mechanisms underlying these differences are unknown. Therefore, we infected a highly susceptible mouse strain (DBA/2J) and a resistant strain (C57BL/6J) with influenza A H1N1 (PR8) and performed genome-wide expression analysis. We found genes expressed in lung epithelium that were specifically down-regulated in DBA/2J mice, whereas a cluster of genes on chromosome 3 was only down-regulated in C57BL/6J. In both mouse strains, chemokines, cytokines and interferon-response genes were up-regulated, indicating that the main innate immune defense pathways were activated. However, many immune response genes were up-regulated in DBA/2J much stronger than in C57BL/6J, and several immune response genes were exclusively regulated in DBA/2J. Thus, susceptible DBA/2J mice showed a hyper-inflammatory response. This response is similar to infections with highly pathogenic influenza virus and may serve as a paradigm for a hyper-inflammatory host response to influenza A virus.     

Neutrophils sustain effective CD8(+) T-cell responses in the respiratory tract following influenza infection

Neutrophils have an important role in early host protection during influenza A virus infection. Their ability to modulate the virus-specific adaptive immune response is less clear. Here, we have used a mouse model to examine the impact of neutrophils on CD8(+) T-cell responses during influenza virus infection. CD8(+) T-cell priming, expansion, migration, cytokine secretion and cytotoxic capacity were investigated in the virus-infected airways and secondary lymphoid organs. To do this, we utilised a Ly6G-specific monoclonal antibody (mAb; 1A8) that specifically depletes neutrophils in vivo. Neutrophil depletion early after infection with influenza virus strain HKx31 (H3N2) did not alter influenza virus-derived antigen presentation or na?ve CD8(+) T-cell expansion in the secondary lymphoid organs. Trafficking of virus-specific CD8(+) T cells into the infected pulmonary airways was also unaltered. Instead, early neutropenia reduced both the overall magnitude of influenza virus-specific CD8(+) T cells, together with impaired cytokine production and cytotoxic effector function. Therefore, neutrophils are important participants in anti-viral mechanisms that sustain effective CD8(+) T-cell responses in the respiratory tract of influenza virus-infected mice.     

Cmv1-independent antiviral role of NK cells revealed in murine cytomegalovirus-infected New Zealand White mice

Ly49H(+) NK cells play a critical role in innate antiviral immune responses to murine CMV (MCMV). Ly49H(b6) recognition of MCMV-encoded m157 on infected cells activates natural killing required for host resistance. We show that mAb 3D10 (anti-Ly49H) recognizes comparable subsets of NK cells from New Zealand White (NZW), New Zealand Black (NZB), and C57BL/6 spleens. However, virus levels in the spleens of MCMV-infected NZW and NZB mice differed greatly. We found that MCMV replication in infected NZW spleens was limited through NK cells. Alternately, NZB mice were profoundly susceptible to MCMV infection. Although 3D10 mAb injections given before infection interfere with Cmv1-type resistance in C57BL/6 mice, similar mAb injections did not affect NZW resistance, likely because NZW NK cell receptors did not bind MCMV-encoded m157. Instead, anti-MCMV host defenses in hybrid NZ offspring were associated with multiple chromosome locations including several putative quantitative trait loci that did not overlap with H-2 or NK gene complex loci. This study revealed a novel pathway used by NK cells to defend against MCMV infection. Thus, the importance of Ly49H in MCMV infection may be shaped by other additional background genes.     

Proinflammatory Cytokine Response and Viral Replication in Mouse Bone Marrow Derived Macrophages Infected with Influenza H1N1 and H5N1 Viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.     

禽流感H5N1亚型病毒对五个不同品系小鼠致病性的比较(英文)

目的比较了不同遗传背景小鼠对禽流感H5N1亚型病毒的致病敏感性,为H5N1禽流感模型制作和机理研究提供依据。方法近交系BALB/c、C57BL/6和封闭群ICR、NIHSwiss和KMSwiss共五个不同品系小鼠。每个品系实验动物30只,分接毒组20只,空白对照组10只,每组雌雄各半。病毒株为A/Goose/Guangdong/NH/2003(H5N1),经测定TCID50为10-4.875/mL。接毒组通过鼻腔接种0.1mL病毒液,对照组接种正常鸡胚尿囊液。小鼠接毒后连续观察14d,观察记录临床症状、体温、体重变化,对在实验期间死亡和实验14d结束后仍然存活的小鼠均进行组织器官病理取材,进行RT-PCR病毒分离检测、HE染色及H5N1抗原特异性免疫组化染色。结果①临床症状:H5N1禽流感病毒能感染五个品系的小鼠,引起呼吸急促等症状和一过性体重、体温下降。②死亡情况:小鼠在接毒后第1天即出现死亡,死亡的高峰期集中在接毒后第3～6天。五个品系小鼠死亡率存在差异,BALB/c为70%,ICR为50%,NIHSwiss为40%,C57BL/6为25%,KMSwiss为10%;③病毒分离:各组接毒小鼠在死亡后均进行了病毒分离,死亡小鼠的肺脏均分离到病毒,其他脏器未分离到病毒。④病理变化:实验期间五个品系死亡小鼠肺脏病理改变相近。大体观:死亡小鼠肺部淤血,呈暗红色,体积增大,局部肺组织实变。镜下观:死亡小鼠的共同病理改变为间质性肺炎,具体表现为肺泡腔及间质出血、炎性细胞浸润;间质增生,肺泡隔增宽;肺泡腔中见纤维素性渗出,透明膜形成。⑤免疫组化结果 :在死亡小鼠的气管上皮细胞和肺巨噬细胞可观察到H5N1禽流感病毒阳性表达。结论小鼠作为H5N1禽流感病毒模型具有普适性,不同品系小鼠感染鹅源H5N1禽流感病毒的临床症状、病程和病理变化与人禽流感病例相似。不同品系小鼠的死亡比例有明显差别,可以根据不同的实验目的 ,选择不同品系的小鼠制作H5N1禽流感动物模型。不同品系的遗传特性对禽流感易感性产生明显的影响,遗传背景可能与H5N1禽流感病毒感染应答机理存在联系:BALB/c和C57BL/6均为近交系,其中BALB/c小鼠的品系特征之一表现为干扰素产量低,接种H5N1病毒后表现为高死亡率(70%),而C57BL/6小鼠的干扰素产量高,接种H5N1病毒后表现为低死亡率(25%),提示不同遗传背景小鼠的干扰素水平与H5N1感染致死具相关性。为进一步研究H5N1禽流感病毒易感性相关基因以及其与宿主免疫反应的关系提供了一个研究基础。     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.     

Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis

Most pandemic influenza virus strains undergo adaptation or reassortment before they acquire the ability to cause fatal infections in a new host species. The pathologic changes and tissue tropism during virus adaptation are not fully understood. Here we investigated pathologic changes and tissue tropism by serial lung-to-lung passaging of human influenza virus strain A/Aichi/2/68 (H3N2) in a BALB/c mouse model. Enhanced pulmonary lesions and systemic virus infection were observed during adaptation. Late passage 10 (P10) virus caused extra-pulmonary spread with necrotic and inflammatory lesions in the brain, heart, spleen and intestine of infected animals, in contrast to infection with earlier passage viruses which were restricted to lungs. Non-conservative mutations in the hemagglutinin (Gly218Glu) and non-structural 1 (Asp125Gly) proteins were identified in P10 virus which exhibited high virulence. Virus growth kinetics showed enhanced replication ability of P10 virus in different cell lines. P10 virus also exhibited the ability to bind to erythrocytes of different host species. These results demonstrate extra-pulmonary spread of influenza virus during adaptation with enhanced replication ability in a new host. This mouse adaptation model may provide a basis for understanding cross-species adaptability corresponding to increased virulence of the influenza A virus, a phenomenon of relevance to the emergence of future highly pathogenic strains.     

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

Background

Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo.

Principal Findings

We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.

Conclusion

A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.     

Changes in the phenotypic properties of highly pathogenic influenza A virus of H5N1 subtype induced by N186I and N186T point mutations in hemagglutinin

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

Specific Depletion of Ly6Chi Inflammatory Monocytes Prevents Immunopathology in Experimental Cerebral Malaria

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6C^hi inflammatory monocytes (by anti-CCR2), Ly6G⁺ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C^hi inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ⁺CD8⁺ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C^hi inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C⁺ monocytes in ECM inflammatory processes.