A view and overview on the control of avian influenza outbreaks in poultry: (5-6) Molecular approaches using avian cytokines and RNA-interference

Chicken cytokines such as chicken interferon-alpha (ChIFN-α), chicken interleukins (ChIL) and Toll-like receptors (TLR) are essential components of chicken’s innate immune system which play a vital role against virus infections [18, 24, 31, 36]. An innovative application of ChIFN-α to antagonize avian influenza virus (AIV) infection in poultry through direct oral feeding or drinking water has received more attention than other components [19, 21, 29, 33, 43]. Sekellick et al. [32] showed that up to 60% of investigated AIV population belonged to the highly pathogenic (HPAI) H5N9 subtype were highly sensitive to the inhibitory effects of ChIFN-α. Interestingly, both IFN-sensitive and -resistant clones were obtained after passage of the resistant clones in the presence of IFN which indicated that resistance to ChIFN-α was transient and did not result from stable genetic changes. Xia, et al. [46] cloned the ChIFN-α gene from three different chicken lines and studied their efficacy against H9N2 viruses in-ovo and in-vivo. Up to 70% of in-ovo treated chicken embryos were protected against H9N2 virus infection in dose dependent manner. Moreover, chickens received ChIFN-α by oculonasal inoculation at one day of age were protected from death upon H9N2 virus infection given 24 hours later. Findings of Meng and co-workers [21] showed that oral administration of exogenous ChIFN-α was effective to prevent and treat chickens experimentally infected with an H9N2 virus. It potentially reduced the viral load in trachea and resulted in rapid recovery of the body weight gain. In another study, White Leghorn (WL) chickens received ChIFN-α in drinking water for 14 successive days augmented detectable humoral anti-influenza antibodies after exposure to a low dose of an LPAIV H7N2 infection [19]. Thus, it has been suggested that regular water administration of ChIFN-α can create “super-sentinel” chickens to detect early infections with few amount of LPAIV [19]. Interestingly, ChIFN-α had antiviral activity against H1N1 and H5N9 viruses not only in chicken but also in duck and turkey primary cell cultures indicating a promising use in other avian species [14]. It has been recently found that ChIFN-α is more potent than the ChIFN-β to inactivate H9N2 virus in chicken fibroblast cell line (DF1) [26]. Recently, chIFN-λ demonstrated a remarkable inhibitory activity against HPAI H5N1, HPAI H7N7 and H9N2 viruses in ovo as well as in three to four-week-old chickens [30]. Intramuscular immunization of four-week-old specific-pathogen-free chickens with the melanoma differentiation-associated gene 5 product (chMDA5) increased resistance of chickens to HPAIV H5N1 infection and reduced virus excretion [17].

Furthermore, oral administration of live attenuated Salmonella enterica serovar Typhimurium expressing ChIFN-α alone or in combination with ChIL-18 significantly reduced clinical signs induced by H9N2 virus and decreased the amount of virus load in cloacal swabs and internal organs [27, 28]. Likewise, chicken immunized with a recombinant fowl pox virus (rFPV) vaccine expressing both the HA gene of H9N2 virus and ChIL-18 survived challenge with an H9N2 virus and did not excrete any virus in swab samples and/or internal organs in comparison to non-vaccinated birds [6]. Also, rFPV expressing the H5, H7 and ChIL-18 genes produced significantly higher humoral and cellular mediated immune response and protected specific pathogen free chickens (SPF) and WL chickens against challenge with an HPAIV H5N1. Vaccinated birds had no virus shedding and showed significant increase in body weight gain [22]. So far, efficiency of avian-cytokines to limit AIV infection has not been adequately studied in other avian species. The duck IL-18 and IL-2 genes had been identified and shown to have 85% and 55% nucleotide identity to the chicken equivalents, respectively. Intramuscular inoculations of the duck IL-18 or IL-2 enhanced the humoral immune response of ducks vaccinated with H5N1 or H9N2 inactivated vaccines, respectively [5, 50]. Likewise, the recombinant goose IL-2 strengthens goose humoral immune responses after vaccination using H9N2 inactivated vaccine [49]. The TLR-3, TLR-7 and TLR9 are other promising chicken cytokines derivatives that showed broad-spectrum anti-influenza virus activity in-vitro and in-ovo [13, 34, 44, 45]. Recently, goose TLR7 was found to be identical to their mammalian counterpart and was triggered by H5N1 virus at the early stage of infection of geese [42].

The previous literatures have shown that avian cytokines are not affected by antigenic changes and they have broad spectrum antiviral activities. Nevertheless, the cost of mass production of chicken cytokines is still too high to be applied in large-scale in poultry industry [33]. Moreover, protein stability, host-specificity and labor associated with mass administration of chicken cytokines under field conditions require significant improvement [27]. 

RNAi is a natural phenomenon used by many organisms as a defense mechanism against foreign microbial invasion, including viruses, that able to wreak potential genetic havoc of the susceptible host [35]. Short-interfering RNA (siRNA) is approximately 21–25 nucleotides specific for highly conserved regions of AIV genomes. It effectively mediates the catalytic degradation of complementary viral mRNAs and results in inhibition of a broad spectrum of influenza viruses replication in cell lines, chicken embryos and mice just before or after initiation of an infection [4, 9, 11, 12, 37, 51]. Tompkins and colleagues [40] found that siRNA specific for the NP or PA genes induced full protection of mice against lethal challenge with the HPAI H5N1 and H7N7 subtypes and markedly decreased virus titers in lungs. Likewise, prophylactic use of PA-specific siRNA molecule significantly reduced lung H5N1 virus titers and lethality in infected mice [47]. Moreover, siRNA targeting M2 or NP genes inhibited replication of H5N1 and H9N2 viruses in canine cell line and partially protected mice against HPAV H5N1 [48]. Recently, Jiao and colleagues designed and tested four siRNAs which were to able to inhibit the expression and accumulation of the NS1 protein of an HPAIV H5N1 in human embryonic kidney cell line [15].

In poultry, Li and others [16] showed that the siRNA targeting NP and/or PA genes inhibited protein expression, RNA transcription and multiplication of HPAIV H5N1 in chicken embryo fibroblasts and ECE as well as prevented apoptosis of infected cells. Likewise, chicken cell line transfected with RNAi molecules specific for the NP or PA of AIV showed decrease the levels of NP mRNA and infective titer of an H10N8 quail virus [1]. Also, NP-specific siRNA reduced H5N1 virus replication in cell culture and ECE [51]. Moreover, siRNA molecules targeting the NP, PA and PB1 genes interfered with replication of H1N1 virus in ECE [9].

One of the most advantages of siRNA application in poultry, in contrast to AIV vaccines, that it might not require an intact immune system [3] which is very important particularly in developing countries where a number of immunosuppressive agents are endemic in poultry. In addition, siRNA molecules targeting the highly conserved regions in influenza genome potentially remain effective regardless of the inter- and intra-subtype genetic and antigenic variations of AIV [3, 38]. Moreover, it has also the potential to reduce the emergence of viable resistant variants [7], in this regard combinations of siRNA molecules “cocktail” targeting several genes/regions may be used simultaneously [10, 20]. Furthermore, there is no risk of recombination between siRNA nucleotides and circulating influenza viruses; hence siRNA is complementary to the influenza virus genome [7]. Moreover, the siRNA dose required for inhibition of AIV is very low (sub-nanomoles) [10]. Nevertheless, arise of mutants with the ability to evade the inhibition effect of siRNA are not fully excluded [3]. Unfortunately, there is no stretch of conserved nucleotides in the NA and HA genes sufficient to generate specific siRNA due to extensive variations in these genes among AIV from different species [10]. The siRNA molecules are quickly degraded in-vivo affording a transient short-term protection and multiple-dose is required [1]. None of the siRNAs must share any sequence identity with the host genome to avoid non-specific RNAi-induced gene silencing of the host cells [2, 8, 10, 41]. Delivery vehicle of siRNA to the site of infection is a major constraint [23, 39] remained to be investigated on flock-level in poultry. There is accumulating evidence that siRNA is efficient to inhibit influenza virus replication in-vitro, however in-vivo studies still missing. Research studies focus on mass application of siRNA in poultry as a spray or via drinking water are highly recommended [25]. 

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