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•HPAI H5N1 viruses undergo antigenic changes.•These antigenic changes may lead to loss of protection against poultry vaccines.•The needs to monitor the H5N1 viruses’ susceptibility to vaccine induced immunity.•A lack of serologic HI reactivity may not always mean lack of vaccine protection.•There is a need for challenge studies in vaccinated poultry. Highly pathogenic avian influenza (HPAI) H5N1 virus continues to circulate in poultry in Asia and Africa posing a threat to both public and animal health. Vaccination, used as an adjunct to improved bio-security and stamping-out policies, contributed to protecting poultry in Hong Kong from HPAI H5N1 infection in 2004–2008 although the virus was repeatedly detected in dead wild birds. The detection of clade 2.3.4 H5N1 viruses in poultry markets and a farm in Hong Kong in 2008 raised the question whether this virus has changed to evade protection from the H5 vaccines in use. We tested the efficacy of three commercial vaccines (Nobilis, Poulvac and Harbin Re-5 vaccine) in specific pathogen free white leghorn chickens against a challenge with A/chicken/Hong Kong/8825-2/2008 (clade 2.3.4) isolated from vaccinated poultry in Hong Kong and A/chicken/Hong Kong/782/2009 (clade 2.3.2). Harbin Re5 vaccine provided the best, albeit not complete protection against challenge with the clade 2.3.4 virus. All three vaccines provided good protection from death and significantly reduced virus shedding following challenge with the clade 2.3.2 virus. Only Harbin Re-5 was able to completely protect chickens from virus shedding as well as mortality. Sera from vaccinated chickens had lower geometric hemagglutination inhibition titers against A/chicken/Hong Kong/8825-2/08, as compared to two other clade 2.3.4 and one clade 0 virus. Alignment of amino-acid sequences of the haemagglutinin of A/chicken/Hong Kong/8825-2/08 and the other H5 viruses revealed several mutations in positions including 69, 71, 83, 95, 133,140, 162, 183, 189, 194 and 270 (H5 numbering) which may correlate with loss of vaccine protection. Our results indicated that the tested HPAI H5N1 (2.3.4) virus has undergone antigenic changes that allow it to evade immunity from poultry vaccines. This highlights the need for continued surveillance and monitoring of vaccine induced immunity, with experimental vaccine challenge studies being done where indicated.

Since 2005, highly pathogenic avian influenza A H5N1 viruses have spread from Asia worldwide, infecting poultry, humans and wild birds. Subsequently, global interest in avian influenza (AI) surveillance increased. Mongolia presents an opportunity to study viruses in wild birds because the country has very low densities of domestic poultry and supports large concentrations of migratory water birds. We conducted AI surveillance in Mongolia over two time periods, 2009-2013 and 2016-2018, utilizing environmental fecal sampling. Fresh fecal samples were collected from water bird congregation sites. Hemagglutinin (HA) and neuraminidase (NA) subtypes of positive samples were identified through viral isolation or molecular assays, with pathogenicity determined by HA subtype or sequencing the HA cleavage site. A total of 10,222 samples were collected. Of these, 7,025 fecal samples were collected from 2009 to 2013, and 3,197 fecal samples were collected from 2016 to 2018. Testing revealed 175 (1.7%) positive samples for low-pathogenicity influenza A, including 118 samples from 2009 to 2013 (1.7%) and 57 samples from 2016 to 2018 (1.8%). HA and NA subtyping of all positives identified 11 subtypes of HA and nine subtypes of NA in 29 different combinations. Within periods, viruses were detected more frequently during the fall season than in the early summer. Mongolia's critical wild bird habitat is positioned as a crossroad of multiple migratory flyways. Our work demonstrates the feasibility of using an affordable environmental fecal sampling approach for AI surveillance and contributes to understanding the prevalence and ecology of low-pathogenicity avian influenza viruses in this important location, where birds from multiple flyways mix.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium’s protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV) in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005–2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured) from 7,855 environmental fecal samples (primarily from ducks), or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans), while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV) were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2), or 2009 and 2010 (clade 2.3.2.1); all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study.

The mechanism for the pathogenesis of H5N1 infection in humans remains unclear. This study reveals that cyclooxygenase-2 (COX-2) was strongly induced in H5N1-infected macrophages in vitro and in epithelial cells of lung tissue samples obtained during autopsy of patients who died of H5N1 disease. Novel findings demonstrated that COX-2, along with tumor necrosis factor α and other proinflammatory cytokines were hyperinduced in epithelial cells by secretory factors from H5N1-infected macrophages in vitro. This amplification of the proinflammatory response is rapid, and the effects elicited by the H5N1-triggered proinflammatory cascade are broader than those arising from direct viral infection. Furthermore, selective COX-2 inhibitors suppress the hyperinduction of cytokines in the proinflammatory cascade, indicating a regulatory role for COX-2 in the H5N1-hyperinduced host proinflammatory cascade. These data provide a basis for the possible development of novel therapeutic interventions for the treatment of H5N1 disease, as adjuncts to antiviral drugs.

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.