Evolution of Pathogenicity of Avian Influenza Virus and Control

The genus influenza belongs to the Orthomyxoviridae family, representing viruses that have a single-strand, negative sense, and segmented RNA genome. RNA virus replication is characterized by high mutation rates, short generation time, and high progeny yields. In addition to mutations introduced by their errorprone polymerases without proofreading capacity, influenza viruses generate genomic variations by homologous and non-homologous recombination and reassortment in viruses with segmented genomes. Thus, influenza viruses exist as heterogeneous populations of closely related variants characterized by one or more dominant master nucleotide genome sequence(s). The nature of these “quasi-species” confers a significant adaptation potential through the selection of mutants that can be best suited to a new environment. This selection allows the rapid evolution of influenza viruses by contributing to antigenic variation, pathogenesis and host range shift.

Poultry species are not a natural reservoir for Avian Influenza Virus (AIV) but those are endemic in many wild bird populations. Especially, species in the order Anseriformes harbor a wide variety of antigenic subtypes and are the most important species for the perpetuation of AIVs. In the natural reservoir, the virus is in evolutionary stasis, which is characterized by a low rate of genetic evolution and the relationship between the host and the virus is practically commensal. Also, infected aquatic wild birds generally shed large amounts of virus whilst the humoral immune response in these species is generally weak and transient. In summary, low pathogenic avian influenza viruses (LPAIV) are naturally present with a high diversity of types and sub-types in the wild avifauna, and infect a high diversity of species with an infection that has a low clinical impact. At the contrary, highly pathogenic avian influenza viruses (HPAIV) have a low diversity with epidemics usually involving only one sub-type, and usually affecting a limited number of domestic species with a high clinical impact.

The HA glycoprotein plays an important role in pathogenicity. In order to be infectious, the HA0 precursors need to be cleaved extracellularly in HA1 and HA2 proteins. LPAIVs are cleaved only extracellularly by trypsin-like proteases. Since these proteases are only found in epithelial cells lining the respiratory and intestinal tract, LPAIVs can only replicate in these organs. The hemagglutinin precursor molecules of HPAIVs however, can additionally be cleaved intracellularly by ubiquitous proteases, of which furin is one of the most important ones since it can be found in cells throughout the body. Consequently, HPAIVs can infect a wide range of cell types and infected animals go through a systemic infection characterized by viremia, massive virus replication and cellular damage in multiple cycles. Additionally, the series of mutations leading towards the insertion of multiple basic amino acids at the cleavage site of H5/H7 LPAIVs is thought to occur only in poultry species. It is thus believed that every HPAI outbreak can be linked to an introduction and subsequent circulation of a LPAIV from the wild bird reservoir to poultry. HPAIVs have only been observed among the H5 and H7 antigenic subtypes. However, pathogenicity of the AI viruses is a polygenic trait which is not only dependent of the HA gene.

In 2003, the highly pathogenic avian influenza (HPAI) H5N1 strain, starting circulating in Asia in 1996, became enzootic in poultry. Indeed, from December 2003 to April 2005, HPAI H5N1 caused outbreaks of avian disease in nine Asian countries. This unprecedented spread of HPAI was associated with a failure of surveillance and control measures in these countries, allowing the spread of the virus to Middle East, then Europe in the summer of 2005, and later to Africa. Historically, geographic barriers appeared to limit the spread of low-pathogenicity avian influenza viruses (LPAIV) through migratory aquatic birds between the Old and New Worlds, allowing distinct lineages of Eurasian and North American viruses to evolve, but such barriers were not complete, as occasional spillovers of gene segments occurred. Phylogenetic analysis and understanding of waterfowl migration patterns suggest that the Eurasian H5N8 clade 2.3.4.4 avian influenza virus emerged in late 2013 in China, spread in early 2014 to South Korea and Japan, and reached Siberia and Beringia by summer 2014 via migratory birds.

Three genetically distinct subgroups emerged and subsequently spread along different flyways during fall 2014 into Europe, North America, and East Asia, respectively. All three subgroups reappeared in Japan, a wintering site for waterfowl from Eurasia and parts of North America. Another unprecedented feature of this large H5N1 HPAI outbreak is its association with human disease and mortality. There was no report of human death due to avian influenza (AI) before the H5N1 avian flu alert in 1997 in Hong Kong, where 6 out of 18 human cases were fatal. The total number of laboratory confirmed human cases since 2003 now reaches 846, including 449 mortalities (53%) at the date of writing. The risk of generation of a new pandemic strain, either by reassortment with circulating human influenza or by direct adaptation to humans remains a threat for public health. Unexpected infection of wild felines, cats and even dogs further illustrates the unusual cross species transmission potential of this H5N1 outbreak. But surprisingly, the last human pandemic was caused by the emergence of a new H1N1 strain, containing genetic segments from pigs. More recently, all influenza paradigms were once again breached by the transmission of an avian LPAI H7N9 in humans. Similarly, since its 1st incursion in 2012, HPAI H7N3 has been reported yearly in Mexico and on March 2016, 4 new HPAI H7N3 outbreaks affecting 3 commercial and 1 backyard farms were reported in 2 states. Recent results indicate that the Mexico HP H7N3 originated from the large North America LPAIV pool through
complicated reassortment events. Different segments were contributed by wild waterfowl from different flyways. Five of the eight segments (HA, NA, NP, M, NS) were introduced from wild birds migrating along the central North American flyway, and PB2, PB1 and PA were introduced via the western North American flyway, highlighting the potential role for Mexico as a hotspot of virus reassortment. Fortunately, spill-over from poultry to wild birds -or even to humans- has not occurred so far in the New World as in the Old one.

Due to the high contagiousness and the extreme severity of the disease, HPAI is the only ‘‘flu’’ of domestic animals considered as epizootic requiring drastic measures such as eradication for control. It has been estimated that hundreds of millions of birds have been culled so far in attempt to control the spread of the Asiatic H5N1 virus. Although the total number of poultry affected by this HPAI still represents a small percentage of the total world poultry production (more than 20 billion per year), the continuing endemic situation of H5N1 in parts of Asia and the spread to other continents are a matter of great concern to the international community. Developed countries have
established surveillance programs for AI in wild birds and poultry. Such surveillance is indispensable for early detection before control and eradication measures can come into motion.

As viral diseases represent the dominant pathology in poultry production, besides biosecurity and surveillance, vaccination remains in most situations the only way of controlling them. The aim of vaccination is to protect the bird against specific diseases, to block pathogen transmission and to provide passive immunity to progeny. However, beside their benefits, maternally derived antibody (MDA) presents some burdens by affecting the induction of specific active immune responses by vaccination and this has become a critical factor in the establishment of satisfactory vaccination schedules. Moreover, considering the flock sizes of commercial poultry operations, it has become necessary to develop effective methods of mass vaccination. Therefore, the ideal vaccine should be cheap and easy to administrate, induce very early onset and life-long protection preferably after only one application, be insensitive to MDA and enable the DIVA principle. Such a “silver bullet”, however, does not currently exist for most pathogens, such as for AIV. Disease challenges in every production area will additionally dictate the type of vaccine application best suited for that area. The aim will be to vaccinate a high enough proportion of the birds in the flock, varying according to the infectious agent involved and the current epidemiological situation.

For epizootic diseases (AI-NDV), the goal will be to maximize protection against the infection whereas in many other situations (enzootic diseases like IB or IBD), vaccination may mainly be employed as a means of minimizing the economic impact of a given disease thus rather prevention against clinical disease than against infection. Actually, many conventional vaccines perform very well in controlled conditions but there are more shortcomings considering the field situation. The initial discovery of new and widely applicable immunologic principles with regard to both innate and adaptive (especially mucosal)  immunity as well as innovative approaches for vaccine design and application represent one of the most prominent ambitions for the avian viral vaccines of the future. Evaluation of both mucosal and systemic immune responses induced by infection or vaccination should provide valuable information, which can assist to improve vaccination, for example by using different administration routes or applying more immunogenic formulations. Moreover, the impressive advancement of molecular virology and biotechnology over the last decades has allowed to manipulate most of the avian viruses and to regenerate them at fashion. This allows identifying target genes for attenuation and sites for insertion of foreign genes to use them as (dual) vector vaccines. Although there is still much room for improvement of vaccines against most of the avian viral diseases, much effort during the last decades has concentrated on the development of better avian influenza (AI) vaccines, considering the short-comings in the domain (no live vaccines, antigenic drift…) and the zoonotic risk.

For the efficient design of a vaccine, it is important to know the type of immune response necessary to afford full protection; this corresponds to the so-called immunological correlates of protection. These parameters are used as baselines for the selection of new vaccines. For certain viruses, antibodydependent immunity will be sufficient important while for others, cell-mediated immunity is essential. A protective immune response to vaccination may be due to the production of antibodies (humoral immunity), the action of sensitized T lymphocytes (cellular immunity), or a combination of both. In many cases, a satisfactory protection will be achieved by conventional vaccines but those may have disadvantages or shortcomings in efficacy or safety. These parameters can be used as baselines for the selection of new vaccines.

Moreover, mucosal immunology is increasingly gaining attention as an area of great potential for the development of vaccines. Indeed, mucosal surfaces are the major places of entry of many infectious agents into the body. Mucosae contain several defined lymphoid tissues that respond specifically to invading antigens, and this immune response can be either cellular or humoral (IgA). Nonetheless, mucosal immunology research has been hampered by the difficulty and labor-intensiveness of collecting samples and by the lack of results in poultry. For instance, so far, little data has been available on IgA mucosal responses to viruses in chickens. All this information is necessary to ensure that a given in vitro potency test is also relevant for assessing in vivo efficacy, and to make sure that the relevant immune response is being measured against the adequate antigens. However, there is still a lack of several immune markers in poultry that can be used as correlates of protection like in mammals (i.e. mice and humans).

 

 

Selected recent readings from our group:

Lage Ferreira H., Vangeluwe D., Van Borm, Poncin O., Dumont N., Ozhelvaci O., Munir M., van den Berg T. and Lambrecht B. (2015). Differential viral fitness between H1N1 and H3N8 avian influenza viruses isolated from Mallards (Anas platyrhynchos). Avian Diseases. 59(4):498- 507.

Marché S., van den Berg T., Houdart P. & Lambrecht B. (2015). Multiyear serological surveillance of notifiable influenza A viruses into Belgian poultry. Avian Diseases. 59, 4, 543-547.

Rauw F, Palya V, Van Borm S, Welby S, Tatar-Kis T, Gardin Y, Dorsey KM, Aly MM, Hassan MK, Soliman MA, Lambrecht B, van den Berg T. (2011). Further evidence of antigenic drift and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains. Vaccine. 29 (14): 2590-2600.

Rauw F., Anbari S., van den Berg T. & Lambrecht B. (2011). Measurement of systemic and local respiratory cell-mediated immunity after influenza infection in chickens. Vet Immunol Immunopathol. 143(1-2): 27-37.

Rauw F., V. Palya, Y. Gardin, T. Tatar-Kis, K. Moore Dorsey, B. Lambrecht and T. van den Berg. (2012). Efficacy of rHVT-AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains. Avian Diseases. 56, 4(S1): 913-922.

Steensels M., Rauw F., van den Berg T, Gardin Y., Palya V. and Lambrecht B. (2015). Protection afforded by an rHVT-H5 vaccine against the 2014 European highly pathogenic H5N8 avian influenza strain. Avian Diseases. doi: 10.1637/11126-050615.

Van Borm S, Jonges M, Lambrecht B, Koch G, Houdart P, van den Berg T. (2014). Molecular Epidemiological Analysis of the Transboundary Transmission of 2003 Highly Pathogenic Avian Influenza H7N7 Outbreaks between The Netherlands and Belgium. Transbound Emerg Dis. 61(1):86-90.

Van Borm S., T. Rosseel, D. Vangeluwe, F. Vandenbussche, T. van den Berg & B. Lambrecht. (2012). Phylogeographic analysis of avian influenza viruses isolated from Charadriiformes in Belgium confirms intercontinental reassortment in gulls. Arch. Virol. 157(8):1509- 1522.

Van Borm S, Vangeluwe D, Steensels M, Poncin O, van den Berg T, Lambrecht B. (2011). Genetic characterization of low pathogenic H5N1 and cocirculating avian influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008. Avian Pathology 40(6):613-28.

van den Berg T. (2014). Vacunas del futuro: el modelo de las enfermedades viricas in Vacunacion Avicultura, pp 177-209, Servet  Edition, Grupo Asis (ISBN: 978-84-942976-0-1). 

van den Berg, T, Lambrecht, B., Marché, S., Steensels, M., Van Borm, S. & Bublot M. (2007). Influenza vaccines and vaccination strategies in birds. Comparative Immunology, Microbiology and Infectious Diseases. 31, 2-3: 121-165.