Multidrug Resistant 2009 A/H1N1 Influenza Clinical Isolate with a Neuraminidase I223R Mutation Retains Its Virulence and Transmissibility in Ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza

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

Exacerbated Innate Host Response to SARS-CoV in Aged Non-Human Primates

The emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza A H5N1, urges the need for deciphering their pathogenesis to develop new intervention strategies. SARS-CoV infection causes acute lung injury (ALI) that may develop into life-threatening acute respiratory distress syndrome (ARDS) with advanced age correlating positively with adverse disease outcome. The molecular pathways, however, that cause virus-induced ALI/ARDS in aged individuals are ill-defined. Here, we show that SARS-CoV-infected aged macaques develop more severe pathology than young adult animals, even though viral replication levels are similar. Comprehensive genomic analyses indicate that aged macaques have a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, with NF-κB as central player, whereas expression of type I interferon (IFN)-β is reduced. Therapeutic treatment of SARS-CoV-infected aged macaques with type I IFN reduces pathology and diminishes pro-inflammatory gene expression, including interleukin-8 (IL-8) levels, without affecting virus replication in the lungs. Thus, ALI in SARS-CoV-infected aged macaques developed as a result of an exacerbated innate host response. The anti-inflammatory action of type I IFN reveals a potential intervention strategy for virus-induced ALI

Proinflammatory Cytokine Responses in Extra-Respiratory Tissues During Severe Influenza

Severe influenza is often associated with disease manifestations outside the respiratory tract. While proinflammatory cytokines can be detected in the lungs and blood of infected patients, the role of extra-respiratory organs in the production of proinflammatory cytokines is unknown. Here, we show that both 2009 pandemic H1N1 influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus induce expression of tumor necrosis factor α, interleukin-6, and interleukin-8 in the respiratory tract and central nervous system. In addition, H5N1 virus induced cytokines in the heart, pancreas, spleen, liver, and jejunum. Together, these data suggest that extra-respiratory tissues contribute to systemic cytokine responses, which may increase the severity of influenza

Detection of intrathecal antibodies to diagnose enterovirus infections of the central nervous system

Background Enterovirus-D68 (EV-D68) predominantly causes respiratory disease. However, EV-D68 infections also have been associated with central nervous system (CNS) complications, most specifically acute flaccid myelitis (AFM). Diagnosing EV-D68-associated CNS disease is challenging since viral RNA is rarely detected in cerebrospinal fluid (CSF). Objective In order to determine an EV antibody index (AI), we evaluated the value of a commercially available quantitative ELISA to detect EV-specific antibodies in paired CSF and blood. Study design Nine paired CSF and blood samples were obtained from patients with EV-D68-associated AFM or from patients with a confirmed EV-associated CNS disease. EV-specific antibodies were detected using a quantitative ELISA. A Reiber diagram analysis was performed, by which the AI was calculated. Subsequently, EV ELISA results were compared with an EV-D68 virus neutralization test. Results ELISA detected EV-specific antibodies in 1 out of the 3 patients with EV-D68-associated AFM and in 3 out of the 6 patients with confirmed EV-associated CNS disease. In these patients, the AI was indicative for intrathecal antibody production against enterovirus. Assay comparison showed that EV-D68 neutralizing antibody detection increased the sensitivity of EV-D68 antibody detection. Conclusions A quantitative EV IgG ELISA in combination with Reiber diagram analysis and AI-calculation can be used as a diagnostic tool for EV-associated CNS disease, including EV-D68. An EV-D68 specific ELISA will improve the sensitivity of the tool. With the growing awareness that the detection of non-polio enteroviruses needs to be improved, diagnostic laboratories should consider implementation of EV serology

Detection of intrathecal antibodies to diagnose enterovirus infections of the central nervous system

Background: Enterovirus-D68 (EV-D68) predominantly causes respiratory disease. However, EV-D68 infections also have been associated with central nervous system (CNS) complications, most specifically acute flaccid myelitis (AFM). Diagnosing EV-D68-associated CNS disease is challenging since viral RNA is rarely detected in cerebrospinal fluid (CSF). Objective: In order to determine an EV antibody index (AI), we evaluated the value of a commercially available quantitative ELISA to detect EV-specific antibodies in paired CSF and blood. Study design: Nine paired CSF and blood samples were obtained from patients with EV-D68-associated AFM or from patients with a confirmed EV-associated CNS disease. EV-specific antibodies were detected using a quantitative ELISA. A Reiber diagram analysis was performed, by which the AI was calculated. Subsequently, EV ELISA results were compared with an EV-D68 virus neutralization test. Results: ELISA detected EV-specific antibodies in 1 out of the 3 patients with EV-D68-associated AFM and in 3 out of the 6 patients with confirmed EV-associated CNS disease. In these patients, the AI was indicative for intrathecal antibody production against enterovirus. Assay comparison showed that EV-D68 neutralizing antibody detection increased the sensitivity of EV-D68 antibody detection. Conclusions: A quantitative EV IgG ELISA in combination with Reiber diagram analysis and AI-calculation can be used as a diagnostic tool for EV-associated CNS disease, including EV-D68. An EV-D68 specific ELISA will improve the sensitivity of the tool. With the growing awareness that the detection of non-polio enteroviruses needs to be improved, diagnostic laboratories should consider implementation of EV serology