Highly Pathogenic H5N1 Research Articles

Absence of detection of highly pathogenic H5N1 in migratory waterfowl in southern France in 2005–2006

During fall 2005, the rapid and wide spread of highly pathogenic (HP) H5N1 avian influenza viruses (AIV) outside Asia alerted European health authorities. Because of abnormal and recurrent field mortality, wild migratory birds were considered to be the main dispersing agent of the virus at an intercontinental scale. European wintering wetlands, such as the Camargue (Rhône delta, France), are identified as potential hot spots for the risk of introduction and transmission of bird-borne diseases. In this study, we investigated the role of migratory waterbirds (mainly ducks) in the spread of HP H5N1 viruses. We combined molecular analysis of living and freshly killed birds with population surveillance (aerial censuses and death surveillance). We sampled 1345 birds belonging to 17 waterbird species (3 orders) in the Camargue between September 2005 and March 2006. The prevalence of AIV was 1.8%. We did not detect HP H5N1 virus. Population censuses did not reveal any population decreases nor abnormal mortalities. We discuss, in the light of these results, the implication of wild migratory ducks in the arrival of HP H5N1 AIV in Europe.

First introduction of highly pathogenic H5N1 avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe

BackgroundSince 2005 highly pathogenic (HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds. HP H5N1 virus was isolated in Denmark for the first time in March 2006. A total of 44 wild birds were found positive for the HP H5N1 infection. In addition, one case was reported in a backyard poultry flock.ResultsFull-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period. The haemagglutinin gene grouped phylogenetically in H5 clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006. The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins.ConclusionThe first case of HP H5N1 infection of wild and domestic birds in Denmark was experienced in March 2006. This is the first full genome characterisation of HP H5N1 avian influenza A virus in the Nordic countries. The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005. These viruses may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time.

Practical Problems in Controlling H5N1 High Pathogenicity Avian Influenza at Village Level in Vietnam and Introduction of Biosecurity Measures

Practical Problems in Controlling H5N1 High Pathogenicity Avian Influenza at Village Level in Vietnam and Introduction of Biosecurity Measures

Thermal Inactivation of H5N1 High Pathogenicity Avian Influenza Virus in Naturally Infected Chicken Meat

Thermal inactivation of the H5N1 high pathogenicity avian influenza (HPAI) virus strain A/chicken/Korea/ES/2003 (Korea/03) was quantitatively measured in thigh and breast meat harvested from infected chickens. The Korea/03 titers were recorded as the mean embryo infectious dose (EID50) and were 108.0 EID50/g in uncooked thigh samples and 107.5 EID50/g in uncooked breast samples. Survival curves were constructed for Korea/03 in chicken thigh and breast meat at 1°C intervals for temperatures of 57 to 61°C. Although some curves had a slightly biphasic shape, a linear model provided a fair-to-good fit at all temperatures, with R2 values of 0.85 to 0.93. Stepwise linear regression revealed that meat type did not contribute significantly to the regression model and generated a single linear regression equation for z-value calculations and D-value predictions for Korea/03 in both meat types. The z-value and the upper limit of the 95% confidence interval for the z-value were 4.64 and 5.32°C, respectively. From the lowest temperature to the highest, the predicted D-values and the upper limits of their 95% prediction intervals (conservative D-values) for 57 to 61°C were 241.2 and 321.1 s, 146.8 and 195.4 s, 89.3 and 118.9 s, 54.4 and 72.4 s, and 33.1 and 44.0 s. D-values and conservative D-values predicted for higher temperatures were 0.28 and 0.50 s for 70°C and 0.041 and 0.073 s for 73.9°C. Calculations with the conservative D-values predicted that cooking chicken meat according to current U.S. Department of Agriculture Food Safety and Inspection Service time-temperature guidelines will inactivate Korea/03 in a heavily contaminated meat sample, such as those tested in this study, with a large margin of safety.

Practical Problems in Controlling H5N1 High Pathogenicity Avian Influenza at Village Level in Vietnam and Introduction of Biosecurity Measures

After a consultancy mission funded by a nongovernmental organization (NGO), information was collected on the dynamics of avian influenza (AI) infection at the rural level in a Vietnamese province with several ongoing outbreaks. AI outbreaks are frequent at village level due to environmental, ecological, agroecological, physical, social, and cultural factors, the underlying factor being poor hygienic conditions. Viral circulation is facilitated by the interactions of the integrated aquaculture, animal raising, horticulture agroecosystem, which relies in the peculiar integration of aquaculture (ponding), animal activities, and horticulture and by the connections with the live-bird market system. The interactions of these factors determine the complex system in which wild birds interact with domestic birds and in which people are constantly exposed to sources of infection, leading to the association between poverty and AI infection in humans. This experience underlines that despite all efforts by the Vietnamese Government, international institutions, and the NGO sector, awareness of AI at the village level needs to be improved. In turn, the leading institutions and international donors funding projects of technical cooperation aimed at tackling AI in Vietnam should invest in a system based on a deep knowledge of the practical problems of village condition to address AI with an effective approach. On the basis of the data collected during the mission, particularly on rural and semi-intensive poultry rearing systems, proposals that encompass the application of an effective vaccination strategy including backyard flocks coupled with dissemination of relevant information on biosecurity measures have been developed for decision makers.

Principles for Vaccine Protection in Chickens and Domestic Waterfowl against Avian Influenza

The H5N1 highly pathogenic (HP) avian influenza (AI) epizootic began with reports of mortality from China in 1996 and, by June 2005, caused outbreaks of disease in nine additional Asian countries, affecting or resulting in culling of over 200 million birds. Vaccines can be used in programs to prevent, manage, or eradicate AI. However, vaccines should only be used as part of a comprehensive control strategy that also includes biosecurity, quarantine, surveillance and diagnostics, education, and elimination of infected poultry. Potent AI vaccines, when properly used, can prevent disease and death, increase resistance to infection, reduce field virus replication and shedding, and reduce virus transmission, but do not provide "sterilizing immunity" in the field; i.e., vaccination does not completely prevent AI virus replication. Inactivated AI vaccines and a recombinant fowlpox-H5-AI vaccine are licensed and used in various countries. Vaccines have been shown to protect chickens, geese, and ducks from H5 HPAI. The inactivated vaccines prevented disease and mortality in chickens and geese, and reduced the ability of the field virus to replicate in gastrointestinal and respiratory tracts. Although the Asian H5N1 HPAI virus did not cause disease or mortality in ducks, the use of inactivated vaccine did reduce field virus replication in the respiratory and intestinal tracts. The inactivated vaccine protected geese from morbidity and mortality, and reduced challenge virus replication. The recombinant fowlpox-H5-AI vaccine has provided similar protection, but the vaccine is used only in chickens and with the advantage of application at 1 day of age in the hatchery.

Sequence and epitope analysis of surface proteins of avian influenza H5N1 viruses from Asian patients

Increasing cases of human infections with the high pathogenic avian influenza virus H5N1 have raised great concem on potential human flu pandemics caused by H5N1. The two viral surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA) proteins, are major antigens of H5N1. Introducing new mutations on these two proteins is the major strategy used by H5N1 to expand host range and to avoid the recognition of host immune systems. We analyzed the two surface proteins of H5N1 from Asian human patients and identified many new mutation sites, including a few that were unique to certain lethal strains. We also analyzed the distribution of mutations on different epitopes of the two surface proteins. A receptor-binding site that might involve in the determination of host specificity of H5N1 was also found. Results reported here provided information for better understanding of the evolution trend of H5N1 genome in human.

The immunogenicity and efficacy against H5N1 challenge of reverse genetics-derived H5N3 influenza vaccine in ducks and chickens

H5N1 avian influenza viruses are continuing to spread in waterfowl in Eurasia and to threaten the health of avian and mammalian species. The possibility that highly pathogenic (HP) H5N1 avian influenza is now endemic in both domestic and migratory birds in Eurasia makes it unlikely that culling alone will control H5N1 influenza. Because ducks are not uniformly killed by HP H5N1 viruses, they are considered a major contributor to virus spread. Here, we describe a reverse genetics-derived high-growth H5N3 strain containing the modified H5 of A/chicken/Vietnam/C58/04, the N3 of A/duck/Germany/1215/73, and the internal genes of A/PR/8/34. One or two doses of inactivated oil emulsion vaccine containing 0.015 to 1.2 μg of HA protein provide highly efficacious protection against lethal H5N1 challenge in ducks; only the two dose regimen has so far been tested in chickens with high protective efficacy.

Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus

High-pathogenicity (HP) avian influenza (AI) virus of the H5N1 subtype has caused an unprecedented epizootic in birds within nine Asian countries/regions since it was first reported in 1996. Vaccination has emerged as a tool for use in managing the infection in view of future eradication. This study was undertaken to determine whether two divergent H5N2 commercial vaccine strains, one based on a European and the other a North American low-pathogenicity AI virus, could protect chickens against a recent Asian H5N1 HPAI virus. The North American and European vaccine viruses had 84 and 91% deduced amino acid sequence similarity to the HA1 segment of haemagglutinin protein of Indonesia H5N1 HPAI challenge virus, respectively. Both vaccine strains provided complete protection from clinical signs and death. The vaccines reduced the number of chickens infected and shedding virus from the respiratory and intestinal tracts at the peak of virus replication. In addition, the quantity of virus shed was reduced by 10(4) to 10(5) median embryo infectious doses. The use of specific neuraminidase inhibition tests allowed identification of infected chickens within the vaccinated groups. These data indicate that the currently available H5 vaccines of European and North American lineages will protect chickens against the Asian H5N1 HPAI virus and reduce environmental contamination by the H5N1 HPAI virus. They will be an adjunct to biosecurity measures to reduce virus transmission.

Microassay for measuring thermal inactivation of H5N1 high pathogenicity avian influenza virus in naturally infected chicken meat

A precise, reproducible microassay was developed to measure thermal inactivation of high pathogenicity avian influenza (HPAI) virus in chicken meat. Small pieces of breast or thigh meat (0.05 g) from chickens infected with A/chicken/Pennsylvania/1370/1983 (H5N2) (PA/83) or A/chicken/Korea/ES/2003 (H5N1) (Korea/03) HPAI viruses were tested for inactivation in the heating block of a thermocycler. Korea/03 infected thigh and breast meat had higher virus concentrations (10 6.8 and 10 5.6 mean embryo infectious doses [EID 50]/g, respectively) compared to PA/83 infected thigh and breast meat (10 2.8 and 10 2.3 EID 50/g, respectively). The samples were ran through a ramp-up cycle from 25 to 70 °C, and meat samples were removed and examined for virus infectivity at 30, 40, 50, 60 and 70 °C, and after treatment for 1, 5, 10, 30 and 60 s at 70 °C. The reduction in virus infectivity titers was dependent on virus concentration and no HPAI virus was isolated after 1 s of treatment at 70 °C. A change in coloration from pink-tan to white was associated with a loss in recovery of infectious virus. The microassay provided a predictable and reproducible method to measure thermal inactivation of HPAI virus in chicken meat.

Public Health Risk from Avian Influenza Viruses

Since 1997, avian influenza (AI) virus infections in poultry have taken on new significance, with increasing numbers of cases involving bird-to-human transmission and the resulting production of clinically severe and fatal human infections. Such human infections have been sporadic and are caused by H7N7 and H5N1 high-pathogenicity (HP) and H9N2 low-pathogenicity (LP) AI viruses in Europe and Asia. These infections have raised the level of concern by human health agencies for the potential reassortment of influenza virus genes and generation of the next human pandemic influenza A virus. The presence of endemic infections by H5N1 HPAI viruses in poultry in several Asian countries indicates that these viruses will continue to contaminate the environment and be an exposure risk with human transmission and infection. Furthermore, the reports of mammalian infections with H5N1 AI viruses and, in particular, mammal-to-mammal transmission in humans and tigers are unprecedented. However, the subsequent risk for generating a pandemic human strain is unknown. More international funding from both human and animal health agencies for diagnosis or detection and control of AI in Asia is needed. Additional funding for research is needed to understand why and how these AI viruses infect humans and what pandemic risks they pose.

Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001.

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia.

Les grippes aviaires, une menace permanente

The avian influenza virus belongs to the influenza type A genus. Each strain can be sub-typed for its surface proteins : hemagglutinin (HA) and neuraminidase (NA). Aquatic birds represent the natural reservoir of the virus and occasionally transmit it to domestic poultry. The clinical form of the disease depends on the pathotype of the circulating strain. Most infections with low pathogenic (LP) strains are asymptomatic whereas those with highly pathogenic (HP) strains induce fowl plague. HP strains emerge from LP ones by mutations and insertions in the HA gene. Avian influenza can be occasionally transmitted to mammals including human. Evolution of recent HP H5N1 strains toward an increased pathogenicity and a wider host spectrum is worrying, and experts fear that these new strains could give rise to the next human influenza pandemic. Vaccination with inactivated or recombinant vaccines can be used in addition to biosafety measures to contend an outbreak.

Comparative susceptibility of selected avian and mammalian species to a Hong Kong-origin H5N1 high-pathogenicity avian influenza virus.

Seventeen avian species and two mammalian species were intranasally inoculated with the zoonotic A/chicken/Hong Kong/220/97 (chicken/HK) (H5N1) avian influenza (AI) virus in order to ascertain a relative range of susceptible hosts and the pathobiology of the resultant disease. A direct association was demonstrated between viral replication and the severity of disease, with four general gradations being observed among these species. These gradations included the following: 1) widespread dissemination with rapid and high mortality, 2) neurological disease relative to viral neurotropism, 3) asymptomatic infection or only mild transient depression associated with minor viral replication, and 4) absence of disease relative to minimal to no viral replication. This investigation not only demonstrates that the chicken/HK virus could infect multiple avian species, but also that the virulence of the chicken/HK virus varied significantly among avian species, including those species that are members of the same order.

Avian influenza in Hong Kong 1997-2002.

In 1997, a high-pathogenicity H5N1 avian influenza virus caused serious disease in both man and poultry in Hong Kong, China. Eighteen human cases of disease were recorded, six of which were fatal. This unique virus was eliminated through total depopulation of all poultry markets and chicken farms in December 1997. Other outbreaks of high-pathogenicity avian influenza (HPAI) caused by H5N1 viruses occurred in poultry in 2001 and 2002. These H5N1 viruses isolated had different internal gene constellations to those isolated in 1997. No new cases of infection or disease in man due to these or other H5N1 viruses have been reported. This paper provides an overview and chronology of the events in Hong Kong relating to avian influenza, covering the period from March 1997 to March 2002.

Pathogenicity and antigenicity of a new influenza a (H5N1) virus isolated from duck meat

Avian influenza A viruses are the ancestral origin of all human influenza viruses. The outbreak of highly pathogenic (HP) avian H5N1 in Hong Kong in 1997 highlighted the potential of these viruses to infect and cause severe disease in humans. Since 1999, HP H5N1 viruses were isolated several times from domestic poultry in Asia. In 2001, a HP H5N1 virus, A/Duck/Anyang/AVL-1/2001 (Dk/Anyang), was isolated from imported frozen duck meat in Korea. Because of this novel source of HP H5N1 virus isolation, concerns were raised about the potential for human exposure and infection; we therefore compared the Dk/Anyang virus with HP H5N1 viruses isolated from humans in 1997 in terms of antigenicity and pathogenicity for mammals. At high doses, Dk/Anyang virus caused up to 50% mortality in BALB/c mice, was isolated from the brains and lymphoid organs of mice, and caused lymphopenia. Overall Dk/Anyang virus was substantially less pathogenic for mice than the H5N1 virus isolated from a fatal human case in 1997. Likewise, Dk/Anyang virus was apathogenic for ferrets. Dk/Anyang virus was antigenically distinguishable by hemagglutination-inhibition (HI) assay from human H5N1 viruses isolated in 1997 and avian H5N1 viruses isolated in 2001 in Hong Kong. Nevertheless, prior infection with Dk/Anyang virus protected mice from death after secondary infection with HP human H5N1 viruses. These results indicate that compared with HP human H5N1 viruses, Dk/Anyang virus is substantially less pathogenic for mammalian species. Nevertheless, the novel source of isolation of this avian H5N1 virus must be considered when evaluating the potential risk to public health.

Efficacy of Vaccines in Chickens against Highly Pathogenic Hong Kong H5N1 Avian Influenza

In 1997, highly pathogenic (HP) H5N1 avian influenza virus (AIV) caused infections in poultry in Hong Kong and crossed into humans, resulting in a limited number of infections including 18 hospitalized cases and six associated deaths. The unique ability of this, AIV to infect both poultry and people raised a concern for the potential of humans to be biological as well as mechanical vectors of this AIV to poultry. The current study was undertaken to determine if existing vaccines and their technologies could be used during an outbreak to protect poultry. Commercial and experimental inactivated whole H5 AIV and baculovirus-expressed AIV H5 hemagglurinin protein vaccines provided protection from clinical signs and death in chickens after lethal challenge by human-origin HP H5N1 Hong Kong strains 156/97 and 483/97. The commercial and experimental inactivated vaccines had mean protective doses ranging from 0.25 to 0.89, which represents the milligrams of viral protein in the vaccines that provided protection from death in half of the birds. Furthermore, the vaccines reduced the ability of the challenge AIV to replicate in chickens and decreased the recovery of challenge AIV from the enteric and respiratory tracts, but the use of a vaccine will nor totally prevent AI virus replication and shedding. Existing vaccines will protect poultry from mortality and reduce virus replication from the new HP AIV strain that can infect both poultry and humans.