Avian Influenza H5N1 Infection Research Articles

Risk factors for highly pathogenic avian influenza (HPAI) H5N1 infection in backyard chicken farms, Thailand

To reduce the risk of highly pathogenic avian influenza (HPAI) H5N1 infection in humans, the pathways by which HPAI is spread in poultry must be determined. Backyard poultry farmers are particularly vulnerable to the threat of HPAI, with both their health and livelihoods at risk. Identifying the risk factors for HPAI infection in backyard farms should allow control measures to be better targeted. To study the risk factors of HPAI H5N1 infection, we carried out a case-control study on backyard chicken farms in Thailand, analyzing 104 case farms and 382 control farms. Data on farming practices and environmental characteristics were analyzed using multivariate logistic regression models. We show that farms where owners bought live chickens from another backyard farm had a higher risk of HPAI H5N1 infection (OR 3.34, 95% CI 1.72–6.47), while those where owners used a disinfectant to clean poultry areas were exposed to lower risk (OR 0.48, 95% CI 0.26–0.87). Our results highlight the important role of the trade of poultry between farms in the transmission of HPAI H5N1, in addition to farming practices and environmental characteristics. Findings from this study may help to tailor prevention measures to the local circumstances of backyard farms in different regions of the world.

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

BackgroundThe mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis.Methodology/Principal FindingsThere are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression.Conclusions/SignificanceThe differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.

Avian influenza H5N1 virus induces cytopathy and proinflammatory cytokine responses in human astrocytic and neuronal cell lines

It has previously been reported that the avian H5N1 type of influenza A virus can be detected in neurons and astrocytes of human brains in autopsy cases. However, the underlying neuropathogenicity remains unexplored. In this study, we used differentiated human astrocytic and neuronal cell lines as models to examine the effect of H5N1 influenza A viral infection on the viral growth kinetics and immune responses of the infected cells. We found that the influenza virus receptors, sialic acid-α2,3-galactose and sialic acid-α2,6-galactose, were expressed on differentiated human astrocytic and neuronal cells. Both types of cells could be infected with H5N1 influenza A viruses, but progeny viruses were only produced from infected astrocytic cells but not neuronal cells. Moreover, increased expression of interleukin (IL)-6 and/or tumor necrosis factor α (TNF-α) mRNA was detected in both astrocytic and neuronal cells at 6 and 24 h post-infection. To examine the biological consequences of such enhanced cytokine expression, differentiated astrocytic and neuronal cells were directly treated with these two cytokines. TNF-α treatment induced apoptosis, as well as proinflammatory cytokine, chemokine and inflammatory responses in differentiated astrocytic and neuronal cells. Taken together, our findings reveal that avian influenza H5N1 viruses can infect human astrocytic and neuronal cells, resulting in the induction of direct cellular damage and proinflammatory cytokine cascades. Our observations suggest that avian influenza H5N1 infection can trigger profound CNS injury, which may play an important role in the influenza viral pathogenesis.

Evaluation of Rapid Antigen Detection Kits for the Diagnosis of Highly Pathogenic Avian Influenza H5N1 Infection

Early detection of highly pathogenic (HP) strains of avian influenza, especially the HP H5N1, is important in terms of controlling and minimizing the spread of the virus. Several rapid antigen detection kits that are able to detect influenza A viruses in less than 1 hr are commercially available, but only a few of them have been evaluated. In this study, four commercially available rapid tests for veterinary usage and two tests for human usage were evaluated and compared. The evaluation of the detection limits of the different tests established with serial dilution of HP H5N1 indicated that most of them have a detection limit of about 10(5) to 10(6) 50% tissue culture infectious dose/ml. None of the tests was able to detect virus in oral and cloacal swabs 24 hr post-experimental infection of specific-pathogen-free chickens with HP H5N1. However, 48 hr postinfection, almost all of the rapid tests were able to detect infected birds (dead or alive). Moreover, organs were also successful samples for detection of H5N1 with the rapid tests. Unexpectedly, the specificity was not very high for some tests. However, in general in this study, the tests for veterinary usage showed better sensitivity. To conclude, these tests offer good indicative value in the event of an outbreak, but as a result of their low sensitivity and some aspecific reactions, test results always need to be confirmed by other methods.

Evaluation of Rapid Antigen Detection Kits for the Diagnosis of Highly Pathogenic Avian Influenza H5N1 Infection

Evaluation of Rapid Antigen Detection Kits for the Diagnosis of Highly Pathogenic Avian Influenza H5N1 Infection

Chicken Faeces Garden Fertilizer: Possible Source of Human Avian Influenza H5N1 Infection

Avian influenza H5N1 infection in humans is typically associated with close contact with infected poultry or other infected avian species. We report on human cases of H5N1 infection in Indonesia where exposure to H5N1-infected animals could not be established, but where the investigation found chicken faeces contaminated with viable H5N1 virus in the garden fertilizer. Human cases of avian influenza H5N1 warrant extensive investigations to determine likely sources of illness and to minimize risk to others. Authorities should regulate the sale and transportation of chicken faeces as fertilizer from areas where H5N1 outbreaks are reported.

Intranasal Flu Vaccine Protective against Seasonal and H5N1 Avian Influenza Infections

BackgroundInfluenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that γ-ray inactivated flu virus can induce cross-reactive Tc cell responses.Methodology/Principal FindingHere, we report that intranasal administration of purified γ-ray inactivated human influenza A virus preparations (γ-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of γ-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections.Conclusions/SignificanceIntranasal γ-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.

Analysis of H5N1 avian influenza infections from wild bird surveillance in Hong Kong from January 2006 to October 2007

Intensive surveillance of dead wild birds for H5N1 avian influenza infection is conducted in Hong Kong. Between January 2006 and October 2007 pooled cloacal and tracheal swabs from 17692 dead wild birds (from 16 different orders including 82 genera) were tested and 33 H5N1 highly pathogenic avian influenza viruses were isolated. No H5N1 infection has occurred in poultry farms since January 2003, or in live poultry markets in Hong Kong since November 2003 until a recent detection of H5N1 virus by surveillance of live poultry markets in June 2008. The gross and histopathology in the various H5N1-infected avian species is described, along with the performance of the virus isolation and polymerase chain reaction (PCR) tests used. This evaluation also included determination of virus titres and detection limits for the H5 haemagglutinin gene (H5)and matrix gene real-time reverse-transcription PCR tests in cloacal and tracheal swabs from 12 wild birds. The viruses isolated belonged to Clades 2.3.2 and 2.3.4, and within Clade 2.3.4 some clustering was evident based on H5 haemagglutinin gene haemagglutinating sequencing. There were no differences in the pathology findings between these subgroupings and the various diagnostic tests gave similar results for these viruses, except for a loss in sensitivity of the H5 real-time reverse-transcription PCR for several viruses in one cluster from birds submitted in February 2007. The use of multiple test methods was important in maintaining the diagnostic sensitivity for detecting avian influenza viruses with high genetic variability.

Exhaled Air and Aerosolized Droplet Dispersion During Application of a Jet Nebulizer

BackgroundAs part of our influenza pandemic preparedness, we studied the dispersion distances of exhaled air and aerosolized droplets during application of a jet nebulizer to a human patient simulator (HPS) programmed at normal lung condition and different severities of lung injury.MethodsThe experiments were conducted in a hospital isolation room with a pressure of − 5 Pa. Airflow was marked with intrapulmonary smoke. The jet nebulizer was driven by air at a constant flow rate of 6 L/min, with the mask reservoir filled with sterile water and attached to the HPS via a nebulizer mask. The exhaled leakage jet plume was revealed by a laser light sheet and images captured by high-definition video. Smoke concentration in the plume was estimated from the light scattered by smoke and droplet particles.FindingsThe maximum dispersion distance of smoke particles through the nebulizer side vent was 0.45 m lateral to the HPS at normal lung condition (oxygen consumption, 200 mL/min; lung compliance, 70 mL/cm H2O), but it increased to 0.54 m in mild lung injury (oxygen consumption, 300 mL/min; lung compliance, 35 mL/cm H2O), and beyond 0.8 m in severe lung injury (oxygen consumption, 500 mL/min; lung compliance, 10 mL/cm H2O). More extensive leakage through the side vents of the nebulizer mask was noted with more severe lung injury.InterpretationHealth-care workers should take extra protective precaution within at least 0.8 m from patients with febrile respiratory illness of unknown etiology receiving treatment via a jet nebulizer even in an isolation room with negative pressure.

Identifying areas of Australia at risk for H5N1 avian influenza infection from exposure to nomadic waterfowl moving throughout the Australo-Papuan region

Since 2003, highly pathogenic avian influenza (HPAI) due to the H5N1 virus has been reported from both domestic poultry and wild birds in over 60 countries and this has resulted in the direct death or slaughter of over 250 million birds. The potential for HPAI to be introduced to Australian commercial poultry via migratory shorebirds returning from Asia has previously been assessed as a low risk. However, introduction of HPAI from areas to the immediate north of Australia via nomadic waterfowl that range throughout the Australo-Papuan region provides a second potential pathway of entry. Surveillance programmes provide an important early warning for Australia's estimated 2,000 commercial poultry farms but to be efficient they should be risk-based and target resources at those areas and sectors of the industry at higher risk of exposure. In order to address this need, this study compared the distribution and movement patterns of native waterfowl to identify regions where the likelihood of HPAI incursion and establishment was highest. Analysis of bird banding records provided information on the maximum distances moved and dispersal patterns of the species of waterfowl of interest. Introduction via Cape York was found to be most likely and all poultry farms in Queensland were found to be within range of waterfowl that can shed H5N1 virus for up to 17 days. The final analysis showed that the area at greatest risk of HPAI introduction is the Atherton tableland near Cairns.

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Correction: Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

98 Hyper-induction of COX-2-medicated proinflammatory cascade: A mechanism for the pathogenesis of avian influenza H5N1 infection

98 Hyper-induction of COX-2-medicated proinflammatory cascade: A mechanism for the pathogenesis of avian influenza H5N1 infection

Hyperinduction of Cyclooxygenase‐2–Mediated Proinflammatory Cascade: A Mechanism for the Pathogenesis of Avian Influenza H5N1 Infection

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 alpha 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.

Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

BackgroundSustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.Methodology / Principal FindingsThe ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 µg DNA given twice either by intramuscular needle injection or with a needle-free device.Conclusions/SignificanceDNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.

The Induction of Proinflammatory Cytokines in Response to Avian Influenza H5N1 Infections and their Role in Pathogenesis and the Enhancement of Virulence

Avian H5N1 influenza viruses first emerged as members of a new human influenza A subtype in Hong Kong in 1997 and there are continuing concerns that members of the subtype will be the cause of the next pandemic. Human-tohuman transmission is rare, but mortality rates associated with avian-to-human transmission are significantly higher than for current human epidemic strains. Severe human H5N1 infections are associated with severe viral pneumonia, diffuse alveolar damage and reactive hemophagocytic syndrome (RHS). A characteristic of RHS, and one of the key determinants of H5N1-mediated pathology, is dysfunctional cytokine production resulting in a cytokine storm. Factors contributing to cytokine dysregulation include viral load, apoptosis and the intrinsic properties of H5N1 virus strains. Here, we review the cytokine response to influenza and try to address the relationship between hypercytokinemia and severe H5N1 pathology. Keywords: Pathogenic, H5N1, cytokine, storm, reactive, hematophagocytic, syndrome

Identifying areas of Australia at risk of H5N1 avian influenza infection from exposure to migratory birds: a spatial analysis

Since 2003, highly pathogenic avian influenza (HPAI) due to H5N1 virus has been reported from both domestic poultry and wild birds in 60 countries resulting in the direct death or slaughter of over 250,000,000 birds. The potential exists for HPAI to spread to Australia via migratory shorebirds returning from Asia with the most likely pathway of introduction into commercial poultry flocks involving the transfer of HPAI from migrating shorebirds to native waterfowl species that subsequently interact with poultry on low security poultry farms. Surveillance programmes provide an important early-warning for Australia's estimated 2,000 commercial poultry farms but, to be efficient, they should be risk-based and target resources at those areas and sectors of the industry at higher risk of exposure. This study compared the distributions of migratory shorebirds and native waterfowl to identify six regions where the likelihood of exotic HPAI incursion and establishment in native waterfowl is highest. Analysis of bird banding records showed that native waterfowl did not move further than 10 km during the spring breeding season when migratory shorebirds arrived in Australia. Therefore, poultry farms within 10 km of significant shorebird habitat in these six regions of highest comparative risk were identified. The final analysis showed that the estimated risk to Australia is low with only two poultry farms, one at Broome and one at Carnarvon, located in the regions of highest risk.

Epidemiology of H5N1 avian influenza

High pathogenic (HP) H5N1 avian influenza (AI) infection has been reported in domestic poultry, wildlife, and human populations since 1996. Risk of infection is associated with direct contact with infected birds. The mode of H5N1 spread from Asia to Europe, Africa and the Far East is unclear; risk factors such as legal and illegal domestic poultry and exotic bird trade, and migratory bird movements have been documented. Measures used to control disease such as culling, stamping out, cleaning and disinfection, and vaccination have not been successful in eradicating H5N1 in Asia, but have been effective in Europe.

Cell-Based Influenza Vaccines

Human vaccines against influenza have been available for almost 60 years and, until recently, were prepared almost entirely from viruses grown in the allantoic cavity of 9- to 11-day-old embryonated chicken eggs. Manufacture involving eggs is not sufficiently flexible to allow vaccine supplies to be rapidly expanded, especially in the face of an impending pandemic. Other problems may arise from the infections of progenitor flocks that adversely affect egg supplies, and from the manufacturing process itself, where breakdowns in sterility can occur from the occasional contamination of large batches of viral allantoic fluid. In addition, egg-grown viruses exhibit differences in antigenicity from viruses isolated in mammalian cell lines from clinical specimens. These concerns and the probable need for greatly expanded manufacturing capability in the future have been brought into focus in recent years by the limited spread of H5N1 avian influenza infections to humans in several Asian countries. Alternative approaches involving the use of accredited anchorage-dependent and -independent preparations of the African Green monkey kidney (Vero), Madin-Darby canine kidney (MDCK) and other cell lines have been pursued by several manufacturers in recent years. Yields comparable with those obtained in embryonated eggs have been achieved. These improvements have occurred in parallel with newer technologies that allow the growth of cells in newer synthetic media that do not contain animal serum, in order to allay the concerns of regulators about the potential for spread of transmissible spongiform encephalopathies.