Multiple Reassortants of H5N8 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Viruses Detected in South Korea during the Winter of 2020–2021

During October 2020–January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.     

Avian influenza infections in birds – a moving target

Avian influenza (AI) is a complex infection of birds, of which the ecology and epidemiology have undergone substantial changes over the last decade. Avian influenza viruses infecting poultry can be divided into two groups. The very virulent viruses cause highly pathogenic avian influenza (HPAI), with flock mortality as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all H5 and H7 viruses cause HPAI. All other viruses cause a milder, primarily respiratory, disease (low pathogenic avian influenza, LPAI), unless exacerbated by other infections or environmental conditions. Until recently, HPAI viruses were rarely isolated from wild birds, but for LPAI viruses extremely high isolation rates have been recorded in surveillance studies, particularly in feral waterfowl. In recent years, there have been costly outbreaks of HPAI in poultry in Italy, the Netherlands and Canada and in each of these countries millions of birds were slaughtered to bring the outbreaks under control. However, these outbreaks tend to have been overshadowed by the H5N1 HPAI virus, initially isolated in China, that has now spread in poultry and/or wild birds throughout Asia and into Europe and Africa, resulting in the death or culling of hundreds of millions of poultry and posing a significant zoonosis threat. Since the 1990s, AI infections due to two subtypes, LPAI H9N2 and HPAI H5N1, have been widespread in poultry across large areas of the world, resulting in a modified eco‐epidemiology and a zoonotic potential. An extraordinary effort is required to manage these epidemics from both the human and animal health perspectives.     

Pathogenicity and transmissibility of three avian influenza A (H5N6) viruses isolated from wild birds

Since 2013, highly pathogenic H5N6 avian influenza viruses (AIVs) have emerged in poultry and caused sporadic human infections in Asia. The recent discovery of three new avian H5N6 viruses – A/oriental magpie-robin/Guangdong/SW8/2014 (H5N6), A/common moorhen/Guangdong/GZ174/2014 (H5N6) and A/Pallas's sandgrouse/Guangdong/ZH283/2015 (H5N6) – isolated from apparently healthy wild birds in Southern China in 2014–2015 raises great concern for the spread of these highly pathogenic AIVs (HPAIVs) and their potential threat to human and animal health. In our study, we conducted animal experiments and tested their pathogenicity in ducks, chickens and mice. Ducks can carry and shed the H5N6 HPAIVs, but show no ill effects. On the other hand, these H5N6 HPAIVs can efficiently infect, transmit and cause death in chickens. Due to the overlap of habitats, domestic ducks play an important role in circulating AIVs between poultry and wild birds. Our results raise the possibility that wild birds disseminate these H5N6 HPAIVs to poultry along their flyways and thus pose a great threat to the poultry industry. These viruses are also highly pathogenic to mice, suggesting they pose a potential threat to mammals and, thus, public health. One virus isolated in 2015 replicates much more efficiently and is more lethal in these animals than the two other viruses isolated in 2014. It seems that the H5N6 viruses tend to be more lethal as time passes. Therefore, it is necessary to vigilantly monitor H5N6 HPAIVs in wild birds and poultry.     

Highly Pathogenic Avian Influenza Clade 2.3.4.4b Subtype H5N8 Virus Isolated from Mandarin Duck in South Korea, 2020

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck (Aix galericulata) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.     

Genetic Characterization and Pathogenesis of Avian Influenza Virus H7N3 Isolated from Spot-Billed Ducks in South Korea,Early 2019

Low-pathogenicity avian influenza viruses (LPAIV) introduced by migratory birds circulate in wild birds and can be transmitted to poultry. These viruses can mutate to become highly pathogenic avian influenza viruses causing severe disease and death in poultry. In March 2019, an H7N3 avian influenza virus—A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3)—was isolated from spot-billed ducks in South Korea. This study aimed to evaluate the phylogenetic and mutational analysis of this isolate. Molecular analysis revealed that the genes for HA (hemagglutinin) and NA (neuraminidase) of this strain belonged to the Central Asian lineage, whereas genes for other internal proteins such as polymerase basic protein 1 (PB1), PB2, nucleoprotein, polymerase acidic protein, matrix protein, and non-structural protein belonged to that of the Korean lineage. In addition, a monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, and the non-deletion of the stalk region in the NA gene indicated that this isolate was a typical LPAIV. Nucleotide sequence similarity analysis of HA revealed that the highest homology (99.51%) of this isolate is to that of A/common teal/Shanghai/CM1216/2017 (H7N7), and amino acid sequence of NA (99.48%) was closely related to that of A/teal/Egypt/MB-D-487OP/2016 (H7N3). An in vitro propagation of the A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3) virus showed highest (7.38 Log₁₀ TCID₅₀/mL) virus titer at 60 h post-infection, and in experimental mouse lungs, the virus was detected at six days’ post-infection. Our study characterizes genetic mutations, as well as pathogenesis in both in vitro and in vivo model of a new Korea H7N3 viruses in 2019, carrying multiple potential mutations to become highly pathogenic and develop an ability to infect humans; thus, emphasizing the need for routine surveillance of avian influenza viruses in wild birds.     

Pathogenicity of H5N8 High Pathogenicity Avian Influenza Virus in Chickens and Ducks from South Korea in 2020–2021

During the 2020–2021 winter season, an outbreak of clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) virus occurred in South Korea. Here, we evaluated the pathogenicity and transmissibility of A/mandarin duck/Korea/H242/2020 (H5N8) (H242/20(H5N8)) first isolated from this outbreak in specific pathogen-free (SPF) chickens and commercial ducks in comparison with those of A/duck/Korea/HD1/2017(H5N6) (HD1/17(H5N6)) from a previous HPAI outbreak in 2017–2018. In chickens, the 50% chicken lethal dose and mean death time of H242/20(H5N8) group were 10^4.5 EID₅₀ and 4.3 days, respectively, which indicate less virulent than those of HD1/17(H5N6) (10^3.6 EID₅₀ and 2.2 days). Whereas, chickens inoculated with H242/20(H5N8) survived longer and had a higher titer of viral shedding than those inoculated with HD1/17(H5N6), which may increase the risk of viral contamination on farms. All ducks infected with either HPAI virus survived without clinical symptoms. In addition, they exhibited a longer virus shedding period and a higher transmission rate, indicating that ducks may play an important role as a silent carrier of both HPAI viruses. These results suggest that the pathogenic characteristics of HPAI viruses in chickens and ducks need to be considered to effectively control HPAI outbreaks in the field.     

Novel Clade 2.3.4.4b Highly Pathogenic Avian Influenza A H5N8 and H5N5 Viruses in Denmark, 2020

Since late 2020, outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4.4b have emerged in Europe. To investigate the evolutionary history of these viruses, we performed genetic characterization on the first HPAI viruses found in Denmark during the autumn of 2020. H5N8 viruses from 14 wild birds and poultry, as well as one H5N5 virus from a wild bird, were characterized by whole genome sequencing and phylogenetic analysis. The Danish H5N8 viruses were found to be genetically similar to each other and to contemporary European clade 2.3.4.4b H5N8 viruses, while the Danish H5N5 virus was shown to be a unique genotype from the H5N5 viruses that circulated at the same time in Russia, Germany, and Belgium. Genetic analyses of one of the H5N8 viruses revealed the presence of a substitution (PB2-M64T) that is highly conserved in human seasonal influenza A viruses. Our analyses showed that the late 2020 clade 2.3.4.4b HPAI H5N8 viruses were most likely new incursions introduced by migrating birds to overwintering sites in Europe, rather than the result of continued circulation of H5N8 viruses from previous introductions to Europe in 2016/2017 and early 2020.     

Highly Pathogenic Avian Influenza Viruses at the Wild–Domestic Bird Interface in Europe: Future Directions for Research and Surveillance

Highly pathogenic avian influenza (HPAI) outbreaks in wild birds and poultry are no longer a rare phenomenon in Europe. In the past 15 years, HPAI outbreaks—in particular those caused by H5 viruses derived from the A/Goose/Guangdong/1/1996 lineage that emerged in southeast Asia in 1996—have been occuring with increasing frequency in Europe. Between 2005 and 2020, at least ten HPAI H5 incursions were identified in Europe resulting in mass mortalities among poultry and wild birds. Until 2009, the HPAI H5 virus outbreaks in Europe were caused by HPAI H5N1 clade 2.2 viruses, while from 2014 onwards HPAI H5 clade 2.3.4.4 viruses dominated outbreaks, with abundant genetic reassortments yielding subtypes H5N1, H5N2, H5N3, H5N4, H5N5, H5N6 and H5N8. The majority of HPAI H5 virus detections in wild and domestic birds within Europe coincide with southwest/westward fall migration and large local waterbird aggregations during wintering. In this review we provide an overview of HPAI H5 virus epidemiology, ecology and evolution at the interface between poultry and wild birds based on 15 years of avian influenza virus surveillance in Europe, and assess future directions for HPAI virus research and surveillance, including the integration of whole genome sequencing, host identification and avian ecology into risk-based surveillance and analyses.     

Serological analysis of serum samples from humans exposed to avian H7 influenza viruses in Italy between 1999 and 2003

We evaluated the potential for avian-to-human transmission of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) H7N1 and LPAI H7N3 viruses that were responsible for several outbreaks of influenza in poultry in Italy between 1999 and 2003. A serological survey of poultry workers was conducted by use of a combination of methods. Evidence of anti-H7 antibodies was observed in 3.8% of serum samples collected from poultry workers during the period in 2003 when LPAI H7N3 virus was circulating. These findings highlight the need for surveillance in people occupationally exposed to avian influenza viruses, so that they can be monitored for the risk of avian-to-human transmission during outbreaks of avian influenza caused by both LPAI and HPAI viruses.     

Molecular Characterization of Highly Pathogenic Avian Influenza Viruses H5N6 Detected in Denmark in 2018–2019

Beginning in late 2017, highly pathogenic avian influenza (HPAI) H5N6 viruses caused outbreaks in wild birds and poultry in several European countries. H5N6 viruses were detected in 43 wild birds found dead throughout Denmark. Most of the Danish virus-positive dead birds were found in the period from February to April 2018. However, unlike the rest of Europe, sporadic HPAI H5N6-positive dead wild birds were detected in Denmark in July, August, September, and December 2018, with the last positive bird being found in January 2019. HPAI viruses were not detected in active surveillance of apparently healthy wild birds. In this study, we use full genome sequencing and phylogenetic analysis to investigate the wild bird HPAI H5N6 viruses found in Denmark. The Danish viruses were found to be closely related to those of contemporary HPAI H5N6 viruses detected in Europe. Their sequences formed two clusters indicating that at least two or more introductions of H5N6 into Denmark occurred. Notably, all viruses detected in the latter half of 2018 and in 2019 grouped into the same cluster. The H5N6 viruses appeared to have been maintained undetected in the autumn 2018.     

The evolution of H5N1 influenza viruses in ducks in southern China

The pathogenicity of avian H5N1 influenza viruses to mammals has been evolving since the mid-1980s. Here, we demonstrate that H5N1 influenza viruses, isolated from apparently healthy domestic ducks in mainland China from 1999 through 2002, were becoming progressively more pathogenic for mammals, and we present a hypothesis explaining the mechanism of this evolutionary direction. Twenty-one viruses isolated from apparently healthy ducks in southern China from 1999 through 2002 were confirmed to be H5N1 subtype influenza A viruses. These isolates are antigenically similar to A/Goose/Guangdong/1/96 (H5N1) virus, which was the source of the 1997 Hong Kong "bird flu" hemagglutinin gene, and all are highly pathogenic in chickens. The viruses form four pathotypes on the basis of their replication and lethality in mice. There is a clear temporal pattern in the progressively increasing pathogenicity of these isolates in the mammalian model. Five of six H5N1 isolates tested replicated in inoculated ducks and were shed from trachea or cloaca, but none caused disease signs or death. Phylogenetic analysis of the full genome indicated that most of the viruses are reassortants containing the A/Goose/Guangdong/1/96-like hemagglutinin gene and the other genes from unknown Eurasian avian influenza viruses. This study is a characterization of the H5N1 avian influenza viruses recently circulating in ducks in mainland China. Our findings suggest that immediate action is needed to prevent the transmission of highly pathogenic avian influenza viruses from the apparently healthy ducks into chickens or mammalian hosts.     

Protective efficacy of crude virus‐like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy

Background Currently, Asian lineage highly pathogenic avian influenza (HPAI) H5N1 has become widespread across continents. These viruses are persistently circulating among poultry populations in endemic regions, causing huge economic losses, and raising concerns about an H5N1 pandemic. To control HPAI H5N1, effective vaccines for poultry are urgently needed. Objective In this study, we developed HPAI virus‐like particle (VLP) vaccine as a candidate poultry vaccine and evaluated its protective efficacy and possible application for differentiating infected from vaccinated animals (DIVA). Methods Specific pathogen‐free chickens received a single injection of HPAI H5N1 VLP vaccine generated using baculovirus expression vector system. Immunogenicity of VLP vaccines was determined using hemagglutination inhibition (HI), neuraminidase inhibition (NI), and ELISA test. Challenge study was performed to evaluate efficacy of VLP vaccines. Results and Conclusions A single immunization with HPAI H5N1 VLP vaccine induced high levels of HI and NI antibodies and protected chickens from a lethal challenge of wild‐type HPAI H5N1 virus. Viral excretion from the vaccinated and challenged group was strongly reduced compared with a mock‐vaccinated control group. Furthermore, we were able to differentiate VLP‐vaccinated chickens from vaccinated and then infected chickens with a commercial ELISA test kit, which offers a promising strategy for the application of DIVA concept.     

Ducks: The “Trojan Horses” of H5N1 influenza

Abstract Wild ducks are the main reservoir of influenza A viruses that can be transmitted to domestic poultry and mammals, including humans. Of the 16 hemagglutinin (HA) subtypes of influenza A viruses, only the H5 and H7 subtypes cause highly pathogenic (HP) influenza in the natural hosts. Several duck species are naturally resistant to HP Asian H5N1 influenza viruses. These duck species can shed and spread virus from both the respiratory and intestinal tracts while showing few or no disease signs. While the HP Asian H5N1 viruses are 100% lethal for chickens and other gallinaceous poultry, the absence of disease signs in some duck species has led to the concept that ducks are the “Trojan horses” of H5N1 in their surreptitious spread of virus. An important unresolved issue is whether the HP H5N1 viruses are maintained in the wild duck population of the world. Here, we review the ecology and pathobiology of ducks infected with influenza A viruses and ducks’ role in the maintenance and spread of HP H5N1 viruses. We also identify the key questions about the role of ducks that must be resolved in order to understand the emergence and control of pandemic influenza. It is generally accepted that wild duck species can spread HP H5N1 viruses, but there is insufficient evidence to show that ducks maintain these viruses and transfer them from one generation to the next.     

Subclinical Infection and Transmission of Clade 2.3.4.4 H5N6 Highly Pathogenic Avian Influenza Virus in Mandarin Duck (Aix galericulata) and Domestic Pigeon (Columbia livia domestica)

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aix galericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.     

Avian influenza surveillance in wild birds in the European Union in 2006

Background Infections of wild birds with highly pathogenic avian influenza (AI) subtype H5N1 virus were reported for the first time in the European Union in 2006. Objectives To capture epidemiological information on H5N1 HPAI in wild bird populations through large‐scale surveillance and extensive data collection. Methods Records were analysed at bird level to explore the epidemiology of AI with regard to species of wild birds involved, timing and location of infections as well as the applicability of different surveillance types for the detection of infections. Results In total, 120,706 records of birds were sent to the Community Reference Laboratory for analysis. Incidents of H5N1 HPAI in wild birds were detected in 14 EU Member States during 2006. All of these incidents occurred between February and May, with the exception of two single cases during the summer months in Germany and Spain. Conclusions For the detection of H5N1 HPAI virus, passive surveillance of dead or diseased birds appeared the most effective approach, whilst active surveillance offered better detection of low pathogenic avian influenza (LPAI) viruses. No carrier species for H5N1 HPAI virus could be identified and almost all birds infected with H5N1 HPAI virus were either dead or showed clinical signs. A very large number of Mallards (Anas platyrhynchos) were tested in 2006 and while a high proportion of LPAI infections were found in this species, H5N1 HPAI virus was rarely identified in these birds. Orders of species that appeared to be very clinically susceptible to H5N1 HPAI virus were swans, diving ducks, mergansers and grebes, supporting experimental evidence. Surveillance results indicate that H5N1 HPAI virus did not establish itself successfully in the EU wild bird population in 2006.     

Bridging the Local Persistence and Long-Range Dispersal of Highly Pathogenic Avian Influenza Virus (HPAIv): A Case Study of HPAIv-Infected Sedentary and Migratory Wildfowls Inhabiting Infected Premises

The past two decades have seen the emergence of highly pathogenic avian influenza (HPAI) infections that are characterized as extremely contagious, with a high fatality rate in chickens, and humans; this has sparked considerable concerns for global health. Generally, the new variant of the HPAI virus crossed into various countries through wild bird migration, and persisted in the local environment through the interactions between wild and farmed birds. Nevertheless, no studies have found informative cases associated with connecting local persistence and long-range dispersal. During the 2016–2017 HPAI H5N6 epidemic in South Korea, we observed several waterfowls with avian influenza infection under telemetric monitoring. Based on the telemetry records and surveillance data, we conducted a case study to test hypotheses related to the transmission pathway between wild birds and poultry. One sedentary wildfowl naturally infected with HPAI H5N6, which overlapped with the home range of one migratory bird with H5-specific antibody-positive, showed itself to be phylogenetically close to the isolates from a chicken farm located within its habitat. Our study is the first observational study that provides scientific evidence supporting the hypothesis that the HPAI spillover into poultry farms is caused by local persistence in sedentary birds, in addition to its long-range dispersal by sympatric migratory birds.     

Spatiotemporal Dynamics of Highly Pathogenic Avian Influenza Subtype H5N8 in Poultry Farms,South Korea

Highly pathogenic avian influenza (HPAI), a zoonotic disease, is a major threat to humans and poultry health worldwide. In January 2014, HPAI virus subtype H5N8 first infected poultry farms in South Korea, and 393 outbreaks, overall, were reported with enormous economic damage in the poultry industry. We analyzed the spatiotemporal distribution of HPAI H5N8 outbreaks in poultry farms using the global and local spatiotemporal interaction analyses in the first (January to July 2014) and second (September 2014 to June 2015) outbreak waves. The space–time K-function analyses revealed significant interactions within three days and in an over-40 km space–time window between the two study periods. The excess risk attributable value (D₀) was maintained despite the distance in the case of HPAI H5N8 in South Korea. Eleven spatiotemporal clusters were identified, and the results showed that the HPAI introduction was from the southwestern region, and spread to the middle region, in South Korea. This spatiotemporal interaction indicates that the HPAI epidemic in South Korea was mostly characterized by short period transmission, regardless of the distance. This finding supports strict control strategies such as preemptive depopulation, and poultry movement tracking. Further studies are needed to understand HPAI disease transmission patterns.     

Contemporary North American influenza H7 viruses possess human receptor specificity: Implications for virus transmissibility

Avian H7 influenza viruses from both the Eurasian and North American lineage have caused outbreaks in poultry since 2002, with confirmed human infection occurring during outbreaks in The Netherlands, British Columbia, and the United Kingdom. The majority of H7 infections have resulted in self-limiting conjunctivitis, whereas probable human-to-human transmission has been rare. Here, we used glycan microarray technology to determine the receptor-binding preference of Eurasian and North American lineage H7 influenza viruses and their transmissibility in the ferret model. We found that highly pathogenic H7N7 viruses from The Netherlands in 2003 maintained the classic avian-binding preference for α2–3-linked sialic acids (SA) and are not readily transmissible in ferrets, as observed previously for highly pathogenic H5N1 viruses. However, H7N3 viruses isolated from Canada in 2004 and H7N2 viruses from the northeastern United States isolated in 2002–2003 possessed an HA with increased affinity toward α2–6-linked SA, the linkage type found prominently on human tracheal epithelial cells. We identified a low pathogenic H7N2 virus isolated from a man in New York in 2003, A/NY/107/03, which replicated efficiently in the upper respiratory tract of ferrets and was capable of transmission in this species by direct contact. These results indicate that H7 influenza viruses from the North American lineage have acquired sialic acid-binding properties that more closely resemble those of human influenza viruses and have the potential to spread to naïve animals.     

The 1999-2000 avian influenza (H7N1) epidemic in Italy: veterinary and human health implications

From the end of March to the beginning of December 1999, 199 outbreaks of low pathogenicity avian influenza (LPAI) were diagnosed in the Veneto and Lombardia regions, which are located in the northern part of Italy. The virus responsible for the epidemic was characterized as a type A influenza virus of the H7N1 subtype of low pathogenicity. On the 17th of December, highly pathogenic avian influenza (HPAI) was diagnosed in a meat turkey flock in which 100% mortality was observed in 72 h. The infection spread to the industrial poultry population of northern Italy including chickens, guinea-fowl, quail, pheasants, ducks and ostriches for a total of 413 outbreaks. Over 13 million birds were affected by the epidemic, which caused dramatic economic losses to the Italian poultry industry with severe social and economic implications. The possibility of H7 virus transmission to humans in close contact with the outbreaks was evaluated through a serological survey. Seven hundred and fifty nine sera were collected and tested for the detection of anti-H7 antibodies by means of the micro-neutralization (MN) and single radial haemolysis (SRH) tests. All samples resulted negative. A limited number of clinical samples were also collected for attempted virus isolation with negative results. Current European legislation considers LPAI and HPAI as two completely distinct diseases, not contemplating any compulsory eradication policy for LPAI and requiring eradication for HPAI. Evidence collected during the Italian 1999-2000 epidemic indicates that LPAI due to viruses of the H7 subtype may mutate to HPAI, and, therefore, LPAI caused by viruses of the H5 or H7 subtypes must be controlled to avoid the emergence of HPAI. A reconsideration of the current definition of avian influenza adopted by the EU, could possibly be an aid to avoiding devastating epidemics for the poultry industry in Member States.