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Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds. [ABSTRACT FROM AUTHOR]

H5N1 highly pathogenic avian influenza virus (HPAIV) infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3), in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1) 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3. [ABSTRACT FROM AUTHOR]

We made an H1N1 vaccine candidate from a virus library consisting of 144 ( = 16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naïve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptidespecific CD8+ T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8+ T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine. [ABSTRACT FROM AUTHOR]

Pandemic (H1N1) 2009 influenza virus spread throughout the world since most people did not have immunity against the virus. In the post pandemic phase when many humans might possess immunity against the pandemic virus, one of the concerns is infection in immunocompromised people. Therefore, we used an immunosuppressed macaque model to examine pathogenicity of the pandemic (H1N1) 2009 virus under an immunocompromised condition. The virus in nasal samples of immunosuppressed macaques infected with the pandemic (H1N1) 2009 virus was detected longer after infection than was the virus in nasal samples of immunocompetent macaques. As expected, not only virus amounts but also virus propagation sites in the immunosuppressed macaques were larger than those in lungs of the immunocompetent macaques when they were infected with the pandemic virus. Immunosuppressed macaques possessed low levels of immune cells producing cytokines and chemokines, but levels of inflammatory cytokines/chemokine interleukin (IL)-6, IL-18, and monocyte chemotactic protein (MCP)-1 in lungs of the immunosuppressed macaques were higher than those in lungs of the immunocompetent macaques, though the differences were not statistically significant. Therefore, under an immunosuppressive condition, the pandemic influenza (H1N1) 2009 virus might cause more severe morbidity with high cytokine/chemokine production by the host innate immune system than that seen in macaques under the immunocompetent condition. [ABSTRACT FROM AUTHOR]

Highly pathogenic avian influenza viruses (HPAIVs) cause lethal infection in chickens. Severe cases of HPAIV infections have been also reported in mammals, including humans. In both mammals and birds, the relationship between host cytokine response to the infection with HPAIVs and lethal outcome has not been well understood. In the present study, the highly pathogenic avian influenza viruses A/turkey/Italy/4580/1999 (H7N1) (Ty/Italy) and A/chicken/Netherlands/2586/2003 (H7N7) (Ck/NL) and the low pathogenic avian influenza virus (LPAIV) A/chicken/Ibaraki/1/2005 (H5N2) (Ck/Ibaraki) were intranasally inoculated into chickens. Ty/Italy replicated more extensively than Ck/NL in systemic tissues of the chickens, especially in the brain, and induced excessive mRNA expression of inflammatory and antiviral cytokines (IFN-γ, IL-1β, IL-6, and IFN-α) in proportion to its proliferation. Using in situ hybridization, IL-6 mRNA was detected mainly in microglial nodules in the brain of the chickens infected with Ty/Italy. Capillary leakage assessed by Evans blue staining was observed in multiple organs, especially in the brains of the chickens infected with Ty/Italy, and was not observed in those infected with Ck/NL. In contrast, LPAIV caused only local infection in the chickens, with neither apparent cytokine expression nor capillary leakage in any tissue of the chickens. The present results indicate that an excessive cytokine response is induced by rapid and extensive proliferation of HPAIV and causes fatal multiple organ failure in chickens. [ABSTRACT FROM AUTHOR]

Two highly pathogenic avian influenza virus strains, A/duck/Hokkaido/WZ83/2010 (H5N1) (WZ83) and A/duck/Hokkaido/WZ101/2010 (H5N1) (WZ101), which were isolated from wild ducks in Japan, were found to be genetically similar, with only two amino acid differences in their M1 and PB1 proteins at positions 43 and 317, respectively. We found that both WZ83 and WZ101 caused lethal infection in chickens but WZ101 killed them more rapidly than WZ83. Interestingly, ducks experimentally infected with WZ83 showed no or only mild clinical symptoms, whereas WZ101 was highly lethal. We then generated reassortants between these viruses and found that exchange of the M gene segment completely switched the pathogenic phenotype in both chickens and ducks, indicating that the difference in the pathogenicity for these avian species between WZ83 and WZ101 was determined by only a single amino acid in the M1 protein. It was also found that WZ101 showed higher pathogenicity than WZ83 in mice and that WZ83, whose M gene was replaced with that of WZ101, showed higher pathogenicity than wild-type WZ83, although this reassortant virus was not fully pathogenic compared to wild-type WZ101. These results suggest that the amino acid at position 43 of the M1 protein is one of the factors contributing to the pathogenicity of H5N1 highly pathogenic avian influenza viruses in both avian and mammalian hosts.

Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13 h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.