A view and overview on the control of avian influenza outbreaks in poultry: (3-6) The use of antiviral chemotherapy

A virus is “a piece of bad news wrapped in a protein coat”. This description was described by Sir Peter Medawar (1915-1987), Noble Prize 1960, summarizing the structure of a virus as genome encodes protein. The genome of avian influenza virus (AIV) is composed of 8 separate RNA gene segments; each segment encodes 1 to 3 proteins. These proteins can be allocated to three categories: surface proteins (hemagglutinin HA; neuraminidase NA; and matrix protein 2 M2), internal proteins (tripartite polymerase PB2, PB1, PA; nucleoprotein NP; matrix protein 1 M1; and nuclear export protein NEP) and non-structural (NS) proteins (NS1 and PB1-F2). Please refer to http://www.virology.ws/influenza-101/ for diagrammatic illustrations of structure and replication of influenza virus.

After infection of the bird mostly via nasal, ocular and/or oral routes, firstly, the virus uses the host-cell components to be able to replicate and spread. Firstly, the viral HA attaches to the sialic acid receptors on the host cell membrane. Then, the virus enters the host cell into a vesicle or endosome. In order influenza virus to be infectious, the HA protein must be cleaved into HA1 and HA2 parts by cell enzymes, i.e. trypsin cleaves low pathogenic strains (LPAIV) and is secreted in the respiratory and intestinal tracts and furin-like proteases cleave HPAIV and are secreted by all types of cells. (Therefore, LPAIV causes limited infection mostly in the respiratory and digestive tracts, whereas HPAIV induces systemic disease). Thereafter, acidification of the endosome occurred with increasing the hydrogen ions transferred from the cell-cytoplasm to the endosome via M2 channels resulting in release "uncoating" of the viral genome into the infected cell. The viral ribonucleoproteins (each gene segment wrapped in polymerase proteins and nucleoprotein) enter the nucleus where the replication occurs. At this stage the virus again will use the cell replication machinery for its own replication. A methylated cap structure "10-15 nucleotides" will be stolen from the cellular messenger RNA "mRNA" by the viral polymerase (i.e. PB2, PB1 and PA) acting as a primer for the synthesis of complementary RNAs (cRNAs) and viral mRNA in a process called "cap-snatching". While the cRNAs will be used for the synthesis of progeny virion RNAs in the nucleus, translation of viral mRNA to the corresponding proteins occurs in the cytoplasm of the host cell. The virus switches off the innate immune system of the bird through the early expression of the NS1 protein. The translated PB2, PB1, PA and NP are imported back to the nucleus to wrap “encapsidate” the vRNAs, meanwhile the HA and NA are glycosylated in the Golgi apparatus before being transferred to the cell membrane beside the M2 protein. M1 and NEP facilitate the transport of RNPs from the nucleus and then to the cytoplasmic membrane. Packaging of the nascent virions occurs by a help of M1 protein where all viral structures are packed together in a lipid bilayer envelop taken from the host-cell membrane during budding. Finally, release of the progeny virion occurs after removal of sialic acid from the HA by the sialidase activity of the NA protein.

Researchers try actively to develop antivirals targeted different viral proteins and/or each of these stages of replication. Actually, trials using chemotherapeutic agents (e.g. antibiotics) for treatment or prevention of influenza (also in poultry) were done even before discovering life-cycle of the virus [81, 113]. In the last four decades attention was paid to the M2 blockers and neuraminidase inhibitors (NAIs), the commonly used antivirals in control of influenza viruses in human, to be used in eradication of AIV in poultry. 

Amantadine hydrochloride and rimantadine are two M2 blockers which interrupt virus life cycle by blocking the influx of hydrogen ions through the M2 ion-channel protein and prevent uncoating of the virus in infected host-cells [55, 56, 109]. 

Amantadine is one of the cheapest anti-influenza drugs. The prophylactic activity of amantadine in poultry was firstly studied in 1960s by Lang, et al. [63] in experimentally infected turkeys with an HPAIV H5N9 isolated in 1966 from Ontario, Canada. Optimum prophylaxis was obtained only when amantadine was administered in an adequate, uninterrupted and sustained amount from at least 2 days pre-infection to 23 days post-infection. During H5N2 outbreaks in Pennsylvania, USA in early 1980s, one of control proposals was the use of amantadine as a therapeutic and/or prophylactic approach. Under experimental condition, amantadine given in drinking water was efficacious to decrease morbidity, mortality, transmissibility and limit decrease in egg production [8, 122]. Nonetheless, all recovered birds were susceptible to reinfection [7, 63, 122] and subclinical infection was reported in most of treated birds [63]. Importantly, amantadine lost its effectiveness as amantadine-resistant mutants emerged within 2–3 days of treatment and killed all in-contact chickens. Amantadine-resistant strains were irreversible, stable and transmissible with pathogenic potential comparable to the wild-type virus. Even more, the resistant mutants replaced the wild-type virus and became dominant [6, 7, 96]. It is worth pointing out that several subtypes of AIV including the HPAIV H5N1 that currently circulate in both humans and birds around the world are mostly resistant to amantadine [16, 17, 22, 41, 47, 49, 62, 114]. A recent study indicated that the frequency of H5N1 amantadine-resistant variants in humans from 2002 to 2012 was approximately double (62.2%) compared with the number of resistant strains of avian origin (31.6%) [38], which has been also confirmed in-vitro [83, 119]. Since the late 1990s, positive selection of amantadine-resistant HPAI H5N1 viruses in poultry in China has been proven to be increased due to extensive illegal application of the relatively inexpensive amantadine by some farmers to control HPAIV H5N1 (and LPAIV H9N2) infections in chickens [23, 41, 46, 87, 101]. Hence, rapid selection of amantadine-resistant variants threatens the effective use of the drug for control of human influenza epidemics and/or pandemics [117], therefore the extra-label use of amantadine in poultry was banned by all concerned international organizations [20, 123]. 

The second M2 blocker is rimantadine. Because of the unavailability of rimantadine in most countries, its use in poultry is not reported until now in the field. However, Webster et al. [121] mentioned that rimantadine administered in drinking water was efficacious against HPAIV H5N2 infection in experimentally infected chickens. Nonetheless, the emergence of rimantadine-resistant variants was comparable to amantadine. It is worth mentioning that the most recent LPAIV H7N9 virus which is responsible for the lethality of a considerable number of humans in China since early 2013 is resistant to amantadine and rimantadine [19, 39]. 

Neuraminidase protein, also known as sialidase, is a surface glycoprotein of influenza virus which plays a vital role in the release of the progeny virions from sialic acid on the infected cells [35]. When exposed to NAIs, influenza virions aggregate on the host cell surface preventing their release and allow the host immune system to eliminate the virus [26, 77]. So far, there are two main NAIs, oseltamivir (Tamiflu®) and zanamivir (Relenza®) have been licensed for influenza treatment in human in several countries [2]. 

In the early 2000s, oseltamivir was discovered as a potent and selective inhibitor of the NA enzyme of influenza viruses [55]. It is currently the drug of choice for the treatment of influenza virus infections in human and being stockpiled in many countries in anticipation of a pandemic [120]. Generally, AIV including H5N1 are sensitive to oseltamivir [68] and a small number of H5N1 strains isolated from avian and human origin have been reported to exhibit resistance to oseltamivir [25, 28, 38, 42, 57, 65, 76, 107, 127]. Oral application of oseltamivir via drinking water reduced the morbidity, mortality, virus excretion and chicken-to-chicken transmission in HPAIV H5N2 experimentally infected chickens [78]. Oseltamivir was non-toxic for chicken embryos and prevented the replication of an HPAIV H7N1 in inoculated eggs [53]. An effective prophylactic administration of oseltamivir in experimentally infected chickens and ducks with LPAI H9N2 and H6N2 viruses was also reported [66]. Although it is very plausible that oseltamivir-resistance mutants emerge after application in poultry as recently reported in wild mallards that previously exposed to oseltamivir [18, 37], however none of the few studies conducted to evaluate efficacy of oseltamivir in domestic poultry reported emergence of resistant strains. In nature, oseltamivir-resistant H5N1 viruses isolated from domestic and wild birds emerged probably due to spontaneous mutations rather than exposure to oseltamivir [76, 80, 86, 125]. 

Zanamivir is currently approved in 19 countries for the treatment and prophylaxis of human influenza [55]. Although, development of zanamivir-resistance in poultry is rare [75], it is not effective in preventing a severe outcome and chicken-to-chicken transmission of an HPAIV H5N2 in experimental chickens [78]. 

Several chemotherapeutics targeting influenza virus proteins have been designed to target potential vulnerabilities in the life cycle of AIV. The HA, NA, NS1 and polymerase proteins are hotspots for these antivirals in the last decade which has been reviewed in more details elsewhere [15, 27, 31, 44, 67, 91, 97, 124] . So far, none of these studies was done in-vivo in birds.