Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.     

Pathogenicity of H5N1 influenza A viruses isolated in Vietnam between late 2003 and 2005

Since late 2003, highly pathogenic H5N1 influenza A viruses have spread among poultry and wild aquatic birds in Asian countries. Transmission of these viruses to humans can be lethal. Most human cases of infection with H5N1 viruses have occurred in Vietnam. Therefore, to understand the pathogenicity in mammals of these H5N1 viruses, we took viruses isolated from poultry (5 strains) and humans (2 strains) in Vietnam and tested their virulence in mice. The results showed that the H5N1 viruses from humans were pathogenic in mice and that one avian isolate was also pathogenic. These findings suggested that the H5N1 viruses circulating in poultry adapted during replication in humans or that strains pathogenic in mice were transmitted directly to humans.     

Genetic variation of highly pathogenic H5N1 avian influenza viruses in Vietnam shows both species-specific and spatiotemporal associations

Domestic poultry act as a reservoir for persistent H5N1 endemicity in Vietnam, and the circulation of poultry flocks across farms and to market is thought to drive the spatial movement and evolution of avian influenza viruses. Using a dataset of complete or nearly full genomic sequences from highly pathogenic H5N1 avian influenza viruses collected in domestic poultry in Vietnam from 2003 to 2007, we explore potential differences in genetic characteristics according to species of isolation and the spatiotemporal characteristics of the viruses. Clustering algorithms and ANOVA indicate that H5N1 viruses in Vietnam show differences in the amount of genetic change that chicken viruses experience as compared to duck viruses, with duck viruses showing higher rates of molecular evolution on all eight of influenza's gene segments. There also exist distinct patterns of genetic differentiation according to the year in which they were isolated. These findings suggest that genetic evolution of avian influenza viruses is continuous through time but could also be mediated by the species in which the viruses occur, information that has implications for prevention efforts.     

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam.     

Recent developments in avian influenza research: epidemiology and immunoprophylaxis

Influenza A viruses have been isolated from humans, from several other mammalian species and a wide variety of avian species, among which, wild aquatic birds represent the natural hosts of influenza viruses. The majority of the possible combinations of the 15 haemagglutinin (HA) and nine neuraminidase (NA) subtypes recognized have been identified in isolates from domestic and wild birds. Infection of birds can cause a wide range of clinical signs, which may vary according to the host, the virus strain, the host's immune status, the presence of any secondary exacerbating microorganisms and environmental factors. Most infections are inapparent, especially in waterfowl and other wild birds. In contrast, infections caused by viruses of H5 and H7 subtypes can be responsible for devastating epidemics in poultry. Despite the warnings to the poultry industry about these viruses, in 1997 an avian H5N1 influenza virus was directly transmitted from birds to humans in Hong Kong and resulted in 18 confirmed infections, thus strengthening the pandemic threat posed by avian influenza (AI). Indeed, reassortant viruses, harbouring a combination of avian and human viral genomes, have been responsible for major pandemics of human influenza. These considerations warrant the need to continue and broaden efforts in the surveillance of AI. Control programmes have varied from no intervention, as in the case of the occurrence of low pathogenic (LP) AI (LPAI) viruses, to extreme, expensive total quarantine-slaughter programmes carried out to eradicate highly pathogenic (HP) AI (HPAI) viruses. The adoption of a vaccination policy, targeted either to control or to prevent infection in poultry, is generally banned or discouraged. Nevertheless, the need to boost eradication efforts in order to limit further spread of infection and avoid heavy economic losses, and advances in modern vaccine technologies, have prompted a re-evaluation of the potential use of vaccination in poultry as an additional tool in comprehensive disease control strategies. This review presents a synthesis of the most recent research on AI that has contributed to a better understanding of the ecology of the virus and to the development of safe and efficacious vaccines for poultry.     

(Highly pathogenic) avian influenza as a zoonotic agent

Zoonotic agents challenging the world every year afresh are influenza A viruses. In the past, human pandemics caused by influenza A viruses had been occurring periodically. Wild aquatic birds are carriers of the full variety of influenza virus A subtypes, and thus, most probably constitute the natural reservoir of all influenza A viruses. Whereas avian influenza viruses in their natural avian reservoir are generally of low pathogenicity (LPAIV), some have gained virulence by mutation after transmission and adaptation to susceptible gallinaceous poultry. Those so-called highly pathogenic avian influenza viruses (HPAIV) then cause mass die-offs in susceptible birds and lead to tremendous economical losses when poultry is affected. Besides a number of avian influenza virus subtypes that have sporadically infected mammals, the HPAIV H5N1 Asia shows strong zoonotic characteristics and it was transmitted from birds to different mammalian species including humans. Theoretically, pandemic viruses might derive directly from avian influenza viruses or arise after genetic reassortment between viruses of avian and mammalian origin. So far, HPAIV H5N1 already meets two conditions for a pandemic virus: as a new subtype it has been hitherto unseen in the human population and it has infected at least 438 people, and caused severe illness and high lethality in 262 humans to date (August 2009). The acquisition of efficient human-to-human transmission would complete the emergence of a new pandemic virus. Therefore, fighting H5N1 at its source is the prerequisite to reduce pandemic risks posed by this virus. Other influenza viruses regarded as pandemic candidates derive from subtypes H2, H7, and H9 all of which have infected humans in the past. Here, we will give a comprehensive overview on avian influenza viruses in concern to their zoonotic potential.     

Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak

During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures.     

Virological surveillance and phylogenetic analysis of the PB2 genes of influenza viruses isolated from wild water birds flying from their nesting lakes in Siberia to Hokkaido, Japan in autumn

Recent introduction of H5N1 highly pathogenic avian influenza virus (HPAIV) in wild birds from poultry in Eurasia signaled the possibility that this virus may perpetuate in nature. Surveillance of avian influenza especially in migratory birds, therefore, has been conducted to provide information on the viruses brought by them to Hokkaido, Japan, from their nesting lakes in Siberia in autumn. During 2008-2009, 62 influenza viruses of 21 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes were isolated. Up to September 2010, no HPAIV has been found, indicating that H5N1 HPAIV has not perpetuated at least dominantly in the lakes where ducks nest in summer in Siberia. The PB2 genes of 54 influenza viruses out of 283 influenza viruses isolated in Hokkaido in 2000-2009 were phylogenetically analysed. None of the genes showed close relation to those of H5N1 HPAIVs that were detected in wild birds found dead in Eurasia on the way back to their northern territory in spring.     

Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006

Avian influenza H5N1 viruses pose a significant threat to human health because of their ability to infect humans directly. In the paper, three highly pathogenic H5N1 influenza viruses were isolated from three species of migratory birds in Qinghai Province of China in 2006. The analysis of the genome sequences indicated that the three isolates shared high homology with each other (94% to 99%). Three isolates shared a common ancestor and were closest to strains isolated from Qinghai and Siberia in 2005, but distinct from poultry viruses found in Southeast Asia. In experimental infection, all three viruses were highly pathogenic to chickens and mice. The results suggest that highly pathogenic avian influenza H5N1 viruses still exist in the migratory birds and could spread to other regions with wild bird migration.     

Wildlife surveillance associated with an outbreak of lethal H5N2 avian influenza in domestic poultry

Wildlife surveillance was conducted for influenza viruses in conjunction with the 1983-84 lethal H5N2 avian influenza epizootic in domestic poultry in Pennsylvania, New Jersey, Maryland, and Virginia. Virus-isolation attempts made on cloacal and tracheal swabs from 4,466 birds and small rodents within the quarantined areas and 1,511 waterfowl in nearby Maryland yielded only a single H5N2 isolate from a pen-raised chukar in Pennsylvania. Antibodies against hemagglutinin type 5 and/or neuraminidase type 2 were found in 33% of the aquatic birds tested; however, this finding could not be used to confirm previous H5N2 avian influenza virus activity because of the possibility of prior infections with multiple influenza subtypes. The low prevalence of lethal H5N2 avian influenza virus in wild birds and small rodents strongly indicated that these animals were not responsible for dissemination of the disease among poultry farms during the outbreak.     

Avian influenza A H5N1 infections in cats

Although cats had been considered resistant to disease from influenza virus infection, domestic cats and large felids are now known to be naturally und experimentally susceptible to infection with highly pathogenic avian influenza virus H5N1 (HPAIV H5N1). The virus causes systemic infection, lung and liver being the mainly affected organs. Infected cats show fever, depression, dyspnoea, and neurological signs, but subclinical infections have also occurred. Mostly, cats have been infected by direct contact with affected birds, especially by eating raw poultry; transmission from cat to cat may also occur. Little is known about the role of cats in the epidemiology of the virus. So far, no reassortment between avian and mammalian influenza viruses has occurred in cats, but experts fear that cats might give the virus an opportunity to adapt to mammals. This publication gives a review on avian influenza in cats with a focus on practical aspects for veterinarians.     

A review of avian influenza in different bird species

Only type A influenza viruses are known to cause natural infections in birds, but viruses of all 15 haemagglutinin and all nine neuraminidase influenza A subtypes in the majority of possible combinations have been isolated from avian species. Influenza A viruses infecting poultry can be divided into two distinct groups on the basis of their ability to cause disease. The very virulent viruses cause highly pathogenic avian influenza (HPAI), in which mortality may be as high as 100%. These viruses have been restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPAI. All other viruses cause a much milder, primarily respiratory disease, which may be exacerbated by other infections or environmental conditions. Since 1959, primary outbreaks of HPAI in poultry have been reported 17 times (eight since 1990), five in turkeys and 12 in chickens. HPAI viruses are rarely isolated from wild birds, but extremely high isolation rates of viruses of low virulence for poultry have been recorded in surveillance studies, giving overall figures of about 15% for ducks and geese and around 2% for all other species. Influenza viruses have been shown to affect all types of domestic or captive birds in all areas of the world, but the frequency with which primary infections occur in any type of bird depends on the degree of contact there is with feral birds. Secondary spread is usually associated with human involvement, probably by transferring infective faeces from infected to susceptible birds.     

Novel reassortant H5N5 viruses bind to a human-type receptor as a factor in pandemic risk

Highly pathogenic avian influenza A(HPAI) H5N1 viruses pose a serious pandemic threat due to their virulence and high mortality in humans, and their increasingly expanding host range and significant ongoing evolution could enhance their human-to-human transmissibility. Recently, various reassortant viruses were detected in different domestic poultry, with the HA gene derived from the A/goose/Guangdong/1/96-like (Gs/GD-like) lineage and the NA gene from influenza viruses of other subtypes. It is reported that some natural reassortant H5N5 highly pathogenic avian influenza viruses were isolated from poultry in China. And their HA genes were belonged to a new clade 2.3.4.4. We evaluated the receptor binding property and transmissibility in guinea pigs of these reassortant H5N5 HPAIVs. The results showed that these viruses bound to both avian-type (α-2,3) and human-type (α-2,6) receptors. In addition, we found that one of these viruses, 031, not only replicated but also transmitted efficiently in guinea pigs. Therefore, such reassortant influenza viruses may pose a pandemic threat.     

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.     

Influenza vaccines and vaccination strategies in birds

Although it is well accepted that the present Asian H5N1 panzootic is predominantly an animal health problem, the human health implications and the risk of human pandemic have highlighted the need for more information and collaboration in the field of veterinary and human health. H5 and H7 avian influenza (AI) viruses have the unique property of becoming highly pathogenic (HPAI) during circulation in poultry. Therefore, the final objective of poultry vaccination against AI must be eradication of the virus and the disease. Actually, important differences exist in the control of avian and human influenza viruses. Firstly, unlike human vaccines that must be adapted to the circulating strain to provide adequate protection, avian influenza vaccination provides broader protection against HPAI viruses. Secondly, although clinical protection is the primary goal of human vaccines, poultry vaccination must also stop transmission to achieve efficient control of the disease. This paper addresses these differences by reviewing the current and future influenza vaccines and vaccination strategies in birds.     

"Constanze": a trinational project on avian influenza in wild birds at Lake Constance

When highly pathogenic avian influenza H5N1 (HPAI H5N1) arrived at Lake Constance in February 2006, little was known about its ecology and epidemiology in wild birds. In order to prevent virus transmission from wild birds to poultry, the adjacent countries initiated the tri-national, interdisciplinary research program ?Constanze? to investigate avian influenza infections in water birds at Lake Constance. In collaboration with government agencies scientists examined the prevalence of AI virus in the region of Lake Constance for a period of 33 months, compared the effectiveness of different surveillance methods and analysed the migration behaviour of water birds. Although virus introduction from regions as far as the Ural Mountains seemed possible based on the migration behaviour of certain species, no influenza A viruses of the highly pathogenic subtype H5N1 (HPAIV) was found. However, influenza A viruses of different low pathogenic subtypes were isolated in 2.2 % of the sampled birds (swabs). Of the different surveillance methods utilised in the program the sampling of so called sentinel birds was particularly efficient.     

Pathogenicity of H5N8 virus in chickens from Korea in 2014

In 2014, two genetically distinct H5N8 highly pathogenic avian influenza (HPAI) viruses were isolated from poultry and wild birds in Korea. The intravenous pathogenicity indices for the two representative viruses were both 3.0. Mortality of chickens intranasally inoculated with the two H5N8 viruses was 100% with a mean death times of 2.5 and 4.5 days. Mortality rates of the contact groups for the two H5N8 viruses were 33.3% and 66.6%. Our study showed that transmissibility of the novel H5N8 viruses was different from that of previously identified H5N1 HPAI viruses, possibly due to genetic changes.     

Avian influenza: eradication from commercial poultry is still not in sight

Avian influenza viruses are highly infectious micro-organisms that primarily affect birds. Nevertheless, they have also been isolated from a number of mammals, including humans. Avian influenza virus can cause large economic losses to the poultry industry because of its high mortality. Although there are pathogenic variants with a low virulence and which generally cause only mild, if any, clinical symptoms, the subtypes H5 and H7 can mutate from a low to a highly virulent (pathogenic) virus and should be taken into consideration in eradication strategies. The primary source of infection for commercial poultry is direct and indirect contact with wild birds, with waterfowl forming a natural reservoir of the virus. Live-poultry markets, exotic birds, and ostriches also play a significant role in the epidemiology of avian influenza. The secondary transmission (i.e., between poultry farms) of avian influenza virus is attributed primarily to fomites and people. Airborne transmission is also important, and the virus can be spread by aerosol in humans. Diagnostic tests detect viral proteins and genes. Virus-specific antibodies can be traced by serological tests, with virus isolation and identification being complementary procedures. The number of outbreaks of avian influenza seems to be increasing - over the last 5 years outbreaks have been reported in Italy, Hong Kong, Chile, the Netherlands, South Korea, Vietnam, Japan, Thailand, Cambodia, Indonesia, Laos, China, Pakistan, United States of America, Canada, South Africa, and Malaysia. Moreover, a growing number of human cases of avian influenza, in some cases fatal, have paralleled the outbreaks in commercial poultry. There is great concern about the possibility that a new virus subtype with pandemic potential could emerge from these outbreaks. From the perspective of human health, it is essential to eradicate the virus from poultry; however, the large number of small-holdings with poultry, the lack of control experience and resources, and the international scale of transmission and infection make rapid control and long-term prevention of recurrence extremely difficult. In the Western world, the renewed interest in free-range housing carries a threat for future outbreaks. The growing ethical objections to the largescale culling of birds require a different approach to the eradication of avian influenza.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.