Mammalian innate resistance to highly pathogenic avian influenza H5N1 virus infection is mediated through reduced proinflammation and infectious virus release

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans

Systemic virus distribution and host responses in brain and intestine of chickens infected with low pathogenic or high pathogenic avian influenza virus

<p>Abstract</p> <p>Background</p> <p>Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens.</p> <p>Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses.</p> <p>Methods</p> <p>To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains.</p> <p>Results</p> <p>Both H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection.</p> <p>Conclusions</p> <p>Although both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.</p

Role of Position 627 of PB2 and the Multibasic Cleavage Site of the Hemagglutinin in the Virulence of H5N1 Avian Influenza Virus in Chickens and Ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens

Core outcomes in neonatology: Development of a core outcome set for neonatal research

Background Neonatal research evaluates many different outcomes using multiple measures. This can prevent synthesis of trial results in meta-analyses and selected outcomes may not be relevant to former patients, parents and health professionals. Objective To define a core outcome set (COS) for research involving infants receiving neonatal care in a high income setting. Design Outcomes reported in neonatal trials and qualitative studies were systematically reviewed. Stakeholders were recruited for a three-round international Delphi survey. A consensus meeting was held to confirm the final COS, based upon the survey results. Participants Four hundred and fourteen former patients, parents, healthcare professionals and researchers took part in the eDelphi survey; 173 completed all 3 rounds. Sixteen stakeholders participated in the consensus meeting. Results The literature reviews identified 104 outcomes; these were included in round one. Participants proposed ten additional outcomes; 114 outcomes were scored in round two and three. Round one scores showed different stakeholder groups prioritised contrasting outcomes. Twelve outcomes were included in the final COS: survival, sepsis, necrotising enterocolitis, brain injury on imaging, general gross motor ability, general cognitive ability, quality of life, adverse events, visual impairment/blindness, hearing impairment /deafness, retinopathy of prematurity and chronic lung disease/bronchopulmonary dysplasia. 6 Conclusions and relevance A COS for clinical trials and other research studies involving infants receiving neonatal care in a high-income setting has been identified. This COS for neonatology will help standardise outcome selection in clinical trials and ensure these are relevant to those most affected by neonatal care

Evidence of Infection by H5N2 Highly Pathogenic Avian Influenza Viruses in Healthy Wild Waterfowl

The potential existence of a wild bird reservoir for highly pathogenic avian influenza (HPAI) has been recently questioned by the spread and the persisting circulation of H5N1 HPAI viruses, responsible for concurrent outbreaks in migratory and domestic birds over Asia, Europe, and Africa. During a large-scale surveillance programme over Eastern Europe, the Middle East, and Africa, we detected avian influenza viruses of H5N2 subtype with a highly pathogenic (HP) viral genotype in healthy birds of two wild waterfowl species sampled in Nigeria. We monitored the survival and regional movements of one of the infected birds through satellite telemetry, providing a rare evidence of a non-lethal natural infection by an HP viral genotype in wild birds. Phylogenetic analysis of the H5N2 viruses revealed close genetic relationships with H5 viruses of low pathogenicity circulating in Eurasian wild and domestic ducks. In addition, genetic analysis did not reveal known gallinaceous poultry adaptive mutations, suggesting that the emergence of HP strains could have taken place in either wild or domestic ducks or in non-gallinaceous species. The presence of coexisting but genetically distinguishable avian influenza viruses with an HP viral genotype in two cohabiting species of wild waterfowl, with evidence of non-lethal infection at least in one species and without evidence of prior extensive circulation of the virus in domestic poultry, suggest that some strains with a potential high pathogenicity for poultry could be maintained in a community of wild waterfowl

Pathogenesis of highly pathogenic avian influenza A/turkey/Turkey/1/2005 H5N1 in Pekin ducks (Anas platyrhynchos) infected experimentally

Asian H5N1 (hereafter referred to as panzootic H5N1) highly pathogenic avian influenza (HPAI) virus has caused large numbers of deaths in both poultry and wild-bird populations. Recent isolates of this virus have been reported to cause disease and death in commercial ducks, which has not been seen with other HPAI viruses. However, little is known about either the dissemination of this H5N1 within the organs or the cause of death in infected ducks. Nineteen 4-week-old Pekin ducks were infected with 106.7 median egg infectious doses of HPAI A/turkey/Turkey/1/05 (H5N1, clade 2.2) in 0.1ml via the intranasal and intraocular routes. Cloacal and oropharyngeal swabs were taken daily before three animals were selected randomly and killed humanely for postmortem examination, when samples of tissues were taken for real-time reverse transcriptase-polymerase chain reaction, histopathological examination and immunohistochemistry. Clinical signs were first observed 4 days post infection (d.p.i.) and included depression, reluctance to feed, in-coordination and torticollis resulting in the death of all the birds remaining on 5d.p.i. Higher levels of virus shedding were detected from oropharyngeal swabs than from cloacal swabs. Real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry identified peak levels of virus at 2d.p.i. in several organs. In the spleen, lung, kidney, caecal tonsils, breast muscle and thigh muscle the levels were greatly reduced at 3d.p.i. However, the highest viral loads were detected in the heart and brain from 3d.p.i. and coincided with the appearance of clinical signs and death. Our experimental results demonstrate the systemic spread of this HPAI H5N1 virus in Pekin ducks, and the localization of virus in the brain and heart tissue preceding death

Experimental infection of highly pathogenic avian influenza virus H5N1 in black-headed gulls (Chroicocephalus ridibundus)

International audienceHistorically, highly pathogenic avian influenza viruses (HPAIV) rarely resulted in infection or clinical disease in wild birds. However, since 2002, disease and mortality from natural HPAIV H5N1 infection have been observed in wild birds including gulls. We performed an experimental HPAIV H5N1 infection of black-headed gulls (Chroicocephalus ridibundus) to determine their susceptibility to infection and disease from this virus, pattern of viral shedding, clinical signs, pathological changes and viral tissue distribution. We inoculated sixteen black-headed gulls with 1 × 104 median tissue culture infectious dose HPAIV H5N1 (A/turkey/Turkey/1/2005) intratracheally and intraesophageally. Birds were monitored daily until 12 days post inoculation (dpi). Oropharyngeal and cloacal swabs were collected daily to detect viral shedding. Necropsies from birds were performed at 2, 4, 5, 6, 7, and 12 dpi. Sampling from selected tissues was done for histopathology, immunohistochemical detection of viral antigen, PCR, and viral isolation. Our study shows that all inoculated birds were productively infected, developed systemic disease, and had a high morbidity and mortality rate. Virus was detected mainly in the respiratory tract on the first days after inoculation, and then concentrated more in pancreas and central nervous system from 4 dpi onwards. Birds shed infectious virus until 7 dpi from the pharynx and 6 dpi from the cloaca. We conclude that black-headed gulls are highly susceptible to disease with a high mortality rate and are thus more likely to act as sentinel species for the presence of the virus than as long-distance carriers of the virus to new geographical areas