Highly pathogenic avian influenza virus H5N1 infects alveolar macrophages without virus production or excessive TNF-alpha induction

Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.     

Preferential HLA usage in the influenza virus-specific CTL response

To study whether individual HLA class I alleles are used preferentially or equally in human virus-specific CTL responses, the contribution of individual HLA-A and -B alleles to the human influenza virus-specific CTL response was investigated. To this end, PBMC were obtained from three groups of HLA-A and -B identical blood donors and stimulated with influenza virus. In the virus-specific CD8(+) T cell population, the proportion of IFN-gamma- and TNF-alpha-producing cells, restricted by individual HLA-A and -B alleles, was determined using virus-infected C1R cells expressing a single HLA-A or -B allele for restimulation of these cells. In HLA-B*2705- and HLA-B*3501-positive individuals, these alleles were preferentially used in the influenza A virus-specific CTL response, while the contribution of HLA-B*0801 and HLA-A*0101 was minor in these donors. The magnitude of the HLA-B*0801-restricted response was even lower in the presence of HLA-B*2705. C1R cells expressing HLA-B*2705, HLA-A*0101, or HLA-A*0201 were preferentially lysed by virus-specific CD8(+) T cells. In contrast, the CTL response to influenza B virus was mainly directed toward HLA-B*0801-restricted epitopes. Thus, the preferential use of HLA alleles depended on the virus studied.     

Avian influenza a virus in wild birds in highly urbanized areas

Avian influenza virus (AIV) surveillance studies in wild birds are usually conducted in rural areas and nature reserves. Less is known of avian influenza virus prevalence in wild birds located in densely populated urban areas, while these birds are more likely to be in close contact with humans. Influenza virus prevalence was investigated in 6059 wild birds sampled in cities in the Netherlands between 2006 and 2009, and compared with parallel AIV surveillance data from low urbanized areas in the Netherlands. Viral prevalence varied with the level of urbanization, with highest prevalence in low urbanized areas. Within cities virus was detected in 0.5% of birds, while seroprevalence exceeded 50%. Ring recoveries of urban wild birds sampled for virus detection demonstrated that most birds were sighted within the same city, while few were sighted in other cities or migrated up to 2659 km away from the sample location in the Netherlands. Here we show that urban birds were infected with AIVs and that urban birds were not separated completely from populations of long-distance migrants. The latter suggests that wild birds in cities may play a role in the introduction of AIVs into cities. Thus, urban bird populations should not be excluded as a human-animal interface for influenza viruses.     

In Vitro and In Vivo Isolation and Characterization of Duvenhage Virus

A fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization of DUVV in vitro and its passage in BALB/c mice. The virus proved to be neuroinvasive in both juvenile and adult mice, resulting in about 50% lethality upon peripheral infection. Clinical signs in infected mice were those of classical rabies. However, the distribution of viral antigen expression in the brain differed from that of classical rabies virus infection and neither inclusion bodies nor neuronal necrosis were observed. This is the first study to describe the in vitro and in vivo isolation and characterization of DUVV.     

Coreceptor Usage of Human Immunodeficiency Virus Type 2 Primary Isolates and Biological Clones Is Broad and Does Not Correlate with Their Syncytium-Inducing Capacities

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with α- or β-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8⁺ cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.     

Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus

Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

Residues of the human metapneumovirus fusion (F) protein critical for its strain-related fusion phenotype: implications for the virus replication cycle

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891-8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511-1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion.     

Emerging viral infections in a rapidly changing world

Emerging viral infections in both humans and animals have been reported with increased frequency in recent years. Recent advances have been made in our knowledge of some of these, including severe acute respiratory syndrome-associated coronavirus, influenza A virus, human metapneumovirus, West Nile virus and Ebola virus. Research efforts to mitigate their effects have concentrated on improved surveillance and diagnostic capabilities, as well as on the development of vaccines and antiviral agents. More attention needs to be given to the identification of the underlying causes for the emergence of infectious diseases, which are often related to anthropogenic social and environmental changes. Addressing these factors might help to decrease the rate of emergence of infectious diseases and allow the transition to a more sustainable society.     

Hyperferritinaemia in Dengue Virus Infected Patients Is Associated with Immune Activation and Coagulation Disturbances

Background

During a dengue outbreak on the Caribbean island Aruba, highly elevated levels of ferritin were detected in dengue virus infected patients. Ferritin is an acute-phase reactant and hyperferritinaemia is a hallmark of diseases caused by extensive immune activation, such as haemophagocytic lymphohistiocytosis. The aim of this study was to investigate whether hyperferritinaemia in dengue patients was associated with clinical markers of extensive immune activation and coagulation disturbances.

Methodology/Principal Findings

Levels of ferritin, standard laboratory markers, sIL-2R, IL-18 and coagulation and fibrinolytic markers were determined in samples from patients with uncomplicated dengue in Aruba. Levels of ferritin were significantly increased in dengue patients compared to patients with other febrile illnesses. Moreover, levels of ferritin associated significantly with the occurrence of viraemia. Hyperferritinaemia was also significantly associated with thrombocytopenia, elevated liver enzymes and coagulation disturbances. The results were validated in a cohort of dengue virus infected patients in Brazil. In this cohort levels of ferritin and cytokine profiles were determined. Increased levels of ferritin in dengue virus infected patients in Brazil were associated with disease severity and a pro-inflammatory cytokine profile.

Conclusions/Significance

Altogether, we provide evidence that ferritin can be used as a clinical marker to discriminate between dengue and other febrile illnesses. The occurrence of hyperferritinaemia in dengue virus infected patients is indicative for highly active disease resulting in immune activation and coagulation disturbances. Therefore, we recommend that patients with hyperferritinaemia are monitored carefully.